Edwardsiella tarda bacteriophage resistant to disinfectants, compositions and uses thereof
By screening out the disinfectant-resistant Edwardsiella tarda phage RKP-ET25012, the problem of phage activity being affected by disinfectant environments has been solved, enabling effective treatment and prevention of Edwardsiella tarda infection, providing an antibiotic alternative, and reducing water pollution and the risk of drug resistance.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO RUNDA BIOTECH
- Filing Date
- 2026-03-11
- Publication Date
- 2026-06-12
AI Technical Summary
Existing bacteriophages exhibit significantly reduced activity when exposed to disinfectant environments, impacting their effectiveness. Furthermore, the overuse of antibiotics leads to drug resistance and water pollution, and there is a lack of effective antibiotic alternatives.
The disinfectant-resistant Edwardsiella tarda phage RKP-ET25012 was screened out. It has the tolerance and high stability to a variety of disinfectants and can be used to prepare drugs, feed additives and antibacterial agents for the treatment and prevention of aquatic diseases caused by Edwardsiella tarda.
Edwardsiella tarda phage RKP-ET25012 maintains high activity in the face of disinfectants and complex environments, effectively treating and preventing aquatic animal diseases, reducing antibiotic use, and providing a safe route of drug administration in aquaculture.
Smart Images
Figure CN122188945A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, specifically to a disinfectant-resistant Edwardsiella tarda bacteriophage, its composition, and its applications. Background Technology
[0002] Edwardsiella is a Gram-negative, short rod-shaped bacterium that is facultatively anaerobic and widely distributed in both freshwater and marine environments. It can infect various freshwater and marine fish species, posing a significant threat to farmed fish. Edwardsiella tarda, in particular, can cause systemic hemorrhagic septicemia in many freshwater and marine fish species, triggering digestive tract diseases in numerous commercially important fish species and resulting in substantial economic losses for the aquaculture industry.
[0003] Currently, the main method for preventing and treating various diseases caused by Edwardsiella pneumoniae infection in aquatic products is to use traditional antibiotics. However, the overuse of antibiotics not only easily leads to the emergence of drug-resistant pathogens but also pollutes water bodies and aquatic products, posing a significant threat to food safety and human health. Therefore, finding new alternatives to antibiotics has become crucial for the prevention and control of Edwardsiella pneumoniae.
[0004] Bacteriophages are natural enemies of bacteria. Discovered before antibiotics, phage therapy has re-emerged as bacterial resistance becomes increasingly serious. Phage therapy holds great promise as an alternative means of eliminating pathogens.
[0005] However, in practical applications, it has been found that the activity of most bacteriophages is significantly affected when exposed to disinfectant environments, thus affecting the effectiveness of their use.
[0006] Therefore, screening for disinfectant-resistant Edwardsiella tarda bacteriophages is of great significance for the prevention and control of diseases caused by Edwardsiella tarda in aquaculture. Summary of the Invention
[0007] To address the technical problem in existing technologies where disinfectants used in the environment affect the activity of Edwardsiella tarda phage, thus impacting its effectiveness, this invention provides a disinfectant-resistant Edwardsiella tarda phage, RKP-ET25012, its composition, and its applications. This Edwardsiella tarda phage is resistant to seven commonly used disinfectants, exhibits a broad lytic spectrum, and high stability. It can be used not only to prepare drugs for the prevention and treatment of diseases caused by Edwardsiella tarda infection, but also for the preparation of aquatic feed additives, disinfectants and kits, and antibacterial agents for aquatic products. Furthermore, it is safe to use and has no side effects, making it a promising antibiotic alternative that can be used to address infections caused by Edwardsiella tarda and water pollution problems caused by the excessive proliferation of Edwardsiella tarda in water bodies.
[0008] The technical solution of this invention is as follows: On the one hand, a disinfectant-resistant Edwardsiella tarda phage (Edwardsiella) bacteriophage) The bacteriophage was named Edwardsiella tarda phage RKP-ET25012 and was deposited on March 7, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46425.
[0009] Electron microscopy revealed that the bacteriophage had the following characteristics: its head was approximately 33 nm long, 22 nm wide, and 65 nm long. Based on the classification criteria of the Ninth Report of the International Committee on Taxonomy of Viruses (ICTV), the bacteriophage was identified as belonging to the family Longtail Phageidae in the order Tail Phages, and was named RKP-ET25012.
[0010] Preferably, the Edwardsiella tarda phage RKP-ET25012 is resistant to at least one of the following disinfectants: 10% chlorine dioxide disinfectant, 20% glutaraldehyde disinfectant, 10% povidone-iodine disinfectant, 10% hydrogen peroxide disinfectant, 0.5% benzalkonium chloride disinfectant, 75% ethanol disinfectant, and 5% sodium hypochlorite disinfectant. This phage retains its resistance for 10 seconds after exposure to these seven commonly used disinfectants. 7 ~10 8 The titer showed broad tolerance to different types of disinfectants. It exhibited particularly significant resistance to povidone-iodine and ethanol disinfectants.
[0011] The Edwardsiella tarda phage RKP-ET25012 maintained a high titer at 50-80℃, especially at 80℃, where it remained at a titer of 1×10⁻⁶. 8 The above demonstrates good thermal stability and heat resistance.
[0012] Bacteriophages maintain good activity at pH 4-10, and their titer remains at 1×10⁻⁶. 8 The above indicates that it maintains strong activity even in complex acidic and alkaline environments.
[0013] The optimal multiplicity of infection (MOI) for the Edwardsiella tarda phage RKP-ET25012 was 0.1, and the titer at the optimal MOI was 5.7 × 10⁻⁶. 10 .
[0014] On the other hand, the aforementioned disinfectant-resistant Edwardsiella tarda phages are used in the preparation of at least one of the following products: (1) Products that kill or inhibit Edwardsiella tarda; (2) Products for the prevention and / or treatment of aquatic diseases caused by Edwardsiella tarda; (3) Products for the prevention and / or treatment of inflammatory reactions caused by Edwardsiella tarda.
[0015] The aquatic diseases caused by the aforementioned Edwardsiella tarda include digestive tract diseases and / or septicemia in aquatic animals.
[0016] On the other hand, a composition comprising the aforementioned Edwardsiella tarda phage RKP-ET.
[0017] The above composition is at least one of pharmaceutical preparations, feed additives, disinfectants, and antibacterial agents.
[0018] Preferably, the dosage form of the pharmaceutical preparation is any one of solution, emulsion, suspension, powder, gel, granules, or lyophilized preparation, and it is used to prevent and treat Edwardsiella tarda via methods such as immersion, injection, or oral administration. The beneficial effects achieved by this invention are as follows: 1. The Edwardsiella tarda phage RKP-ET25012 of this invention is resistant to seven commonly used environmental disinfectants. It maintains its activity at pH 4-10 and 80°C, and exhibits strong lytic activity against 90% of environmental isolates. It can treat aquatic animal diseases caused by Edwardsiella tarda infection, reduce the overuse of antibiotics, and provide a healthy and safe new approach for aquaculture medication. 2. The Edwardsiella tarda phage RKP-ET25012 in this invention has a good killing effect on Edwardsiella tarda isolated from aquaculture farms. It can be used to prevent and treat diseases caused by Edwardsiella tarda infection, reduce the use of antibiotics, and has good specificity and biosafety. Attached Figure Description
[0019] Figure 1 This is a colony morphology diagram of Edwardsiella tarda provided by the present invention.
[0020] Figure 2 This is a phage pattern of host bacteria by Edwardsiella tarda phage, provided by the present invention.
[0021] Figure 3 This is an electron microscope image of Edwardsiella tarda phage RKP-ET25012, which is the subject of this invention.
[0022] Figure 4 This is a one-step growth curve of the Edwardsiella tarda phage RKP-ET25012 of this invention.
[0023] Figure 5 This is the thermal stability curve of the Edwardsiella tarda phage RKP-ET25012 of the present invention.
[0024] Figure 6 This is the pH stability curve of Edwardsiella tarda phage RKP-ET25012 of the present invention. Detailed Implementation
[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.
[0026] Example 1: Isolation and Identification of Edwardsiella tarda Samples were taken from the diseased farms, and aseptically streaked onto BHI solid medium. After incubation at 28°C for 18 hours, smooth, slightly convex, and round colonies were picked for purification. Figure 1 Then, single colonies were picked and cultured in BHI liquid medium for 12 hours to obtain a bacterial suspension. Using 16S rRNA gene sequencing and serotype identification, it was identified as *Edwardsiella tarda*, and named ET24012.
[0027] Example 2: Phage Isolation and Purification Wastewater was collected from the farm, centrifuged at 10,000 rpm for 5 minutes to remove larger impurities and most bacteria, and then filtered through a 0.22 μm filter. 0.1 mL of the bacterial suspension and 1 mL of the wastewater filtrate were added to 4 mL of LB liquid medium, incubated overnight at 37°C with shaking, and then centrifuged at 10,000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter. The presence of plaques was identified using the double-plate method. A single, independent, uniformly shaped, clear, and transparent plaque was picked from a double-layer plate containing plaques. Figure 2 The purified phage was obtained by repeating the double plate method three times.
[0028] The purified phages were plated on double-layer plates. A single plaque was then placed in LB broth along with host bacterial suspension. After phage proliferation, the proliferation solution was centrifuged at 10,000 rpm for 5 minutes and filtered through a 0.22 μm filter. The filtrate was then serially diluted 100-fold to a final concentration of 10⁻⁶. 8 Take 0.1 ml of the last three dilutions of phage and 0.1 ml of the host bacterial suspension, spread them on double-layer plates, and determine the phage titer.
[0029] Phage titer (pfu / mL) = number of phage plaques × dilution factor ÷ 0.1 The calculated phage titer is approximately 3 × 10⁻⁶. 9 .
[0030] The bacteriophage suspension was mixed with 60% glycerol at a ratio of 1:1 and stored at -80℃, named RKP-ET25012. It was deposited on March 7, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 46425.
[0031] Example 3: Morphological observation of bacteriophages 1. Experimental Method: Take 20 μL of liquid containing crude phage particles and drop it onto a copper grid. Allow it to settle naturally for 15 min. Then, use filter paper to absorb the excess liquid from the side. Add one drop of 2% phosphotungstic acid (PTA) to the copper grid to stain the phage for 10 min. Then, use filter paper to absorb the staining solution from the side. After the sample dries, observe the phage morphology using an electron microscope.
[0032] 2. Experimental Results and Analysis: The results showed that the head of the bacteriophage was about 33 nm long, about 22 nm wide, and about 65 nm long. According to the classification criteria of the 9th report of the International Committee on Taxonomy of Viruses (ICTV), the bacteriophage was identified as belonging to the family Longtail Phageidae in the order Tail Phages, and was named RKP-ET25012.
[0033] Example 4: Molecular biological identification of bacteriophages 1. Experimental Method: Phage whole-genome sequencing and analysis: Libraries were constructed using the Illumina TruSeq™ Nano DNA SamplePrepKit method. Specific steps were as follows: (1) A library was constructed starting with 1 μg of phage genomic DNA; (2) Covaris M220 ultrasound breaks DNA down to 300-500 bp; (3) Fill in the 3' end with A and connect the index adapter (TruSeq™ Nano DNA Sample Prep Kit); (4) Library enrichment, PCR amplification for 8 cycles; (5) 2% agarose gel recovery target band (Certified Low Range Ultra Agarose); (6) TBS380 (Picogreen) quantitative analysis, mixing according to data ratio before loading; (7) Bridged PCR amplification was performed on the cBot solid-phase support to generate clusters; (8) Illumina Hiseq sequencing platform, 2×150bp sequencing was performed.
[0034] 2. Experimental Results and Analysis: Whole-genome sequencing results showed that the genome of phage RDP-ET25012 is a 59324 bp circular double-stranded DNA, with a total of 97 open reading frames (ORFs) predicted. These ORFs cover core processes such as phage assembly, DNA replication and repair, transcriptional regulation, host lysis, and nucleotide metabolism. They contain key functional genes such as perforin, lyase, and DNA polymerase. After comprehensive annotation and verification, no toxin genes, antibiotic resistance genes, or lysogeny-related integrase genes, which pose safety risks, were detected.
[0035] Example 5: Bacteriophage tolerance to disinfectant experiment 1. Experimental Method: Take eight 50 mL sterile centrifuge tubes and add 30 mL of Edwardsiella tarda phage suspension RKP-ET25012 (initial titer 3 × 10⁻⁶) to each tube. 9 The first seven mice were treated with disinfectants in the following order: chlorine dioxide (10%), glutaraldehyde (20%), povidone-iodine (10%), hydrogen peroxide (10%), benzalkonium chloride (0.5%), ethanol (75%), and sodium hypochlorite (5%). The last mouse was treated with physiological saline as a control group. After 1 hour, 1 mL of each solution was collected and the potency was determined using the double-layer plate method. The experiment was repeated three times, and the average value was taken.
[0036] 2. Experimental Results and Analysis: The test results are shown in Table 1. The Edwardsiella tarda phage RKP-ET25012 retained 10 μL of its normal activity after exposure to seven commonly used disinfectants. 7 ~10 8 The titer showed broad tolerance to different types of disinfectants. It exhibited significant resistance, particularly to povidone-iodine and ethanol.
[0037] The above results confirm that this bacteriophage can still effectively lyse the host bacteria in actual breeding environments after routine disinfection with disinfectants, demonstrating good disinfectant resistance and application prospects.
[0038] Table 1. Phage titers after treatment with different disinfectants
[0039] Example 6: Determination of the Multiple of Infection by Bacteriophage 1. Experimental Method: Edwardsiella tarda phage RKP-ET25012 liquid and host bacterial culture were added to LB liquid medium at MOI ratios of 0.001, 0.01, 0.1, 1, and 10. After incubation at 37°C with shaking at 140 rpm for 8 h, the culture was centrifuged at 12000 r / min for 5 min at room temperature. The supernatant was then plated on double-layer plates to determine its potency.
[0040] 2. Experimental Results and Analysis: As shown in Table 2, the optimal multiplicity of infection for the Edwardsiella tarda phage RKP-ET25012 is 0.1, at which point the phage titer is 5.7 × 10⁻⁶. 10 .
[0041] Table 2. Phage titers at different multiplicity of infection
[0042] Example 7: One-step growth curve 1. Experimental Method: Mix 1 mL of host bacteria with 0.1 mL of phage fluid and incubate at 37°C for 15 min. Centrifuge the incubated mixture at 12000 r / min for 5 min, then remove the supernatant and collect the precipitate. Wash the precipitate twice with sterile physiological saline. Add 10 mL of preheated LB liquid medium (37°C) to the precipitate and mix well. Immediately take 1 mL of the mixture as the 0 min sample and incubate it in a shaker at 37°C. Take samples every 10 min to determine the phage titer. Perform three replicates and take the average value. Plot a one-step growth curve with infection time on the x-axis and phage titer on the y-axis.
[0043] Outbreak size = Total number of bacteria at the end of the phage outbreak / Total number of bacteria at the beginning of the phage outbreak.
[0044] 2. Experimental Results and Analysis: The results are as follows Figure 4 As shown, the titer of bacteriophages did not change significantly 30 minutes after infecting the host bacteria, indicating that the incubation period is approximately 30 minutes; the titer of bacteriophages after infecting the host bacteria is approximately 30 minutes. Within 80 minutes, the number of bacteriophages increased dramatically, indicating that the phage outbreak period was about 50 minutes, with an outbreak rate of about 150 PFU / cell. In the following 40 minutes, the number of bacteriophages remained basically unchanged, entering a stable growth phase.
[0045] Example 8: Temperature stability experiment of bacteriophage 1. Experimental Method: One mL of Edwardsiella tarda phage suspension RKP-ET25012 was placed in centrifuge tubes and incubated in constant temperature water baths at 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 30 min, 60 min, and 90 min, respectively. The phage titer was determined using the double-plate method. Double-sample incubation was performed at each point, and the average value was taken. The experiment was repeated three times.
[0046] 2. Experimental Results and Analysis: The results are as follows Figure 5 As shown, the titer of Edwardsiella tarda phage RKP-ET25012 remained at a high level after treatment at 30℃-60℃ for 30 min, 60 min, and 90 min; the titer decreased when the temperature exceeded 70℃; and the phage was almost inactivated after treatment at 90℃ for more than 60 min.
[0047] Example 9: pH stability experiment of bacteriophage 1. Experimental Method: The pH values of LB medium were adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, respectively. The medium was placed in a water bath at 37°C. After the temperature stabilized, 900 μL of the mixture was combined with 100 μL of phage solution, and incubated at 37°C for 60 min. The phage titer was then determined using the double-layer plate method. Double-sample culture was performed at each point, and the average value was taken. The experiment was repeated three times.
[0048] 2. Experimental Results and Analysis: like Figure 6 As shown, bacteriophages can maintain good activity at pH 4-10, and their titer remains at 1×10⁻⁶. 8 In summary, bacteriophages are almost inactivated when pH < 2.0 or pH > 12.0.
[0049] Example 10: Determination of phage lysis profile 1. Experimental Method: Thirty Edwardsiella tarda strains isolated from aquaculture environments were selected. 100 μL of the test bacterial suspension was mixed thoroughly with LB semi-solid medium and poured onto the plates. After drying, 20 μL of Edwardsiella tarda phage RKP-ET25012 was added dropwise to the plate containing the host bacteria. After absorption, the plate was incubated at 37℃ for 6-8 hours. The presence of phage plaques was observed; if plaques appeared, it indicated that the phage had a lytic effect on the strain.
[0050] 2. Experimental Results and Analysis: As shown in Table 3, this bacteriophage exhibited lytic activity against 27 out of 30 strains of Edwardsiella tarda, with a lysis rate of 90%. It demonstrated a good lytic effect on the host and has significant practical application value.
[0051] Table 3: Lysis spectrum of Edwardsiella tarda phage RKP-ET25012
[0052] Note: In the table, "+" indicates cleavage and "-" indicates no cleavage.
[0053] 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 disinfectant-resistant Edwardsiella tarda phage (Edwardsiella bacteriophage) Its features are: The bacteriophage was named Edwardsiella tarda phage RKP-ET25012 and was deposited on March 7, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46425.
2. The disinfectant-resistant Edwardsiella tarda phage according to claim 1, characterized in that: The described Edwardsiella tarda phage RKP-ET25012 is resistant to at least one of the following disinfectants: 10% chlorine dioxide disinfectant, 20% glutaraldehyde disinfectant, 10% povidone-iodine disinfectant, 10% hydrogen peroxide disinfectant, 0.5% benzalkonium chloride disinfectant, 75% ethanol disinfectant, and 5% sodium hypochlorite disinfectant.
3. The disinfectant-resistant Edwardsiella tarda phage according to claim 1, characterized in that: The Edwardsiella tarda phage RKP-ET was treated at 80°C for 30 min, maintaining a titer of 1×10⁻⁶. 8 above.
4. The disinfectant-resistant Edwardsiella tarda phage according to claim 1, characterized in that: The Edwardsiella tarda phage RKP-ET25012 remained active at pH 4-10 and 80°C.
5. The disinfectant-resistant Edwardsiella tarda phage according to claim 1, characterized in that: The optimal multiplicity of infection (MOI) for the Edwardsiella tarda phage RKP-ET25012 was 0.1, and the titer at the optimal MOI was 5.7 × 10⁻⁶. 10 .
6. The use of the disinfectant-resistant Edwardsiella tarda phage according to claim 1 in the preparation of at least one of the following products: (1) Products that kill or inhibit Edwardsiella tarda; (2) Products for the prevention and / or treatment of aquatic diseases caused by Edwardsiella tarda; (3) Products for the prevention and / or treatment of inflammatory reactions caused by Edwardsiella tarda.
7. The application according to claim 6, characterized in that: The aquatic diseases caused by Edwardsiella tarda include digestive tract diseases and / or septicemia in aquatic animals.
8. A composition, characterized in that: It contains the Edwardsiella tarda phage RKP-ET25012 as described in any one of claims 1-5.
9. The composition according to claim 8, characterized in that: The composition is at least one of a pharmaceutical preparation, a feed additive, a disinfectant, and an antibacterial agent.
10. The composition according to claim 9, characterized in that: The dosage form of the drug preparation is any one of solution, emulsion, suspension, powder, gel, granule or lyophilized preparation, and it is used to prevent and treat Edwardsiella tarda by means of immersion, injection or oral administration.