Aeromonas hydrophila bacteriophage vBAhM7 and application thereof

Abstract

The invention provides an aeromonas hydrophila bacteriophage vBAhM7 and application thereof, and belongs to the technical field of microorganisms. According to the invention, aeromonas hydrophila is taken as host bacteria, a bacteriophage vBAhM7 is separated from sewage at a water inlet of a sewage treatment plant, and the titer can reach 109 PFU / mL; the tolerable temperature is 30-70 DEG C, and the high-temperature-resistant property is good; when the pH value is 3-12, the activity is relatively high, and the acid-base tolerance is good; the bacteriophage is not sensitive to chloroform, has no lipid coating on the shell, is stable in structure, and can resist the damage of organic solvents; the inhibition rate range on the host bacteria biofilm is 36.0%-68.6%, and good host bacteria biofilm inhibition capability is achieved; the in-vitro sterilization effect on host bacteria is good, and a relatively strong inhibition effect is achieved; the zebra fish can be protected; the antibacterial agent can be used for preventing and controlling aeromonas hydrophila in aquatic products, and the use of antibacterial agents in aquaculture is avoided or reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a bacteriophage vB_AhM_7 of Aeromonas hydrophila and application thereof. BACKGROUND

[0002] Aeromonas hydrophila belongs to the genus Aeromonas of the family Vibrionaceae and is a gram-negative short rod. Aeromonas hydrophila is a kind of conditional pathogenic bacteria and can cause diseases in humans and animals. Research reports show that the exotoxin produced by Aeromonas hydrophila is an important pathogenic factor. The identified exotoxins include aerolysin, hemolysin and cytolytic enterotoxin. Aeromonas hydrophila can infect a wide range of hosts, such as grass carp, crucian carp, largemouth bass, bream, megalozestrone, tilapia and grouper, and can cause motile aeromonas septicemia and tail rot disease. Bacterial septicemia caused by Aeromonas hydrophila has a wide range of epidemic, a long epidemic time, a high incidence and a high mortality rate, and poses a great threat to aquaculture. The bacteria can directly attack the central nervous system, causing brain tissue damage and permanent nervous system damage. Symptoms of human infection with Aeromonas hydrophila include sudden severe headache, fever, nausea and vomiting. As the disease progresses, the patient's symptoms become more severe and specific, and over time, may cause seizures, hallucinations, loss of consciousness, and even coma. The disease caused by Aeromonas hydrophila progresses very quickly, and if not treated in time, the disease may rapidly worsen within a week of infection, leading to death. Antibacterial drugs are generally used to prevent or treat bacterial diseases in aquaculture, but the irrational use or misuse of antibacterial drugs has led to the emergence of drug-resistant bacteria. The incidence of multiple drug-resistant Aeromonas hydrophila infection is on the rise, posing new challenges to the aquaculture industry and threatening human health and food safety. Therefore, an alternative method for treating Aeromonas hydrophila is urgently needed. Bacteriophages are viruses that attack bacteria and have strict host specificity. Bacteriophages are the most common and widely distributed group of viruses and are easy to isolate and screen. SUMMARY

[0003] Therefore, the present application aims to provide a bacteriophage vB_AhM_7 of Aeromonas hydrophila and application thereof.

[0004] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0005] The application provides an Aeromonas hydrophila phage vB_AhM_7, which is preserved in the Guangdong Microbial Culture Collection Center on June 17, 2025, and has a preservation number of GDMCC No: 66525-B1.

[0006] The application also provides application of the Aeromonas hydrophila phage vB_AhM_7 in inhibiting Aeromonas hydrophila.

[0007] The application also provides application of the Aeromonas hydrophila phage vB_AhM_7 in inhibiting a biofilm of Aeromonas hydrophila.

[0008] The application also provides application of the Aeromonas hydrophila phage vB_AhM_7 in preparing a medicine for preventing and treating diseases caused by Aeromonas hydrophila.

[0009] Preferably, the diseases include gastrointestinal inflammation, wound infection, necrotizing fasciitis, septicemia, tail rot disease, ulcer disease, furuncle disease, enteritis disease and gill rot disease.

[0010] The application also provides application of the Aeromonas hydrophila phage vB_AhM_7 in preparing aquatic feed for preventing and treating diseases caused by Aeromonas hydrophila.

[0011] Preferably, the diseases include septicemia, tail rot disease, ulcer disease, furuncle disease, enteritis disease and gill rot disease.

[0012] Compared with the prior art, the application has the following beneficial effects:

[0013] The application takes Aeromonas hydrophila as a host bacterium, and a strain of Aeromonas hydrophila phage vB_AhM_7 is isolated from sewage at an inlet of a sewage treatment plant, wherein the optimal infection multiple is 0.01, the titer can reach 10 9PFU / mL; the tolerance temperature is 30-70 DEG C, and it has good high temperature resistance; it has high activity at pH 3-12, and has good acid and alkali tolerance; it is not sensitive to chloroform, the phage shell has no lipid envelope, and the structure is stable, and can resist the damage of organic solvents; the inhibition rate of the host bacteria biofilm is in the range of 36.0%-68.6%, and it has good ability to inhibit the host bacteria biofilm; it has good bactericidal effect on the host bacteria in vitro, and has strong inhibition; it has protective effect on zebrafish; it can be used for preventing and controlling Aeromonas hydrophila in aquatic products, avoiding or reducing the use of antibacterial drugs in aquaculture, and has good practical application value and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is the double-layer plate phage plaque morphology diagram of the phage of the present application;

[0015] Figure 2 is the BLAST result of the phage vB_AhM_7 genome sequence in GenBank (as of June 30, 2025);

[0016] Figure 3 is the ANI comparison result of the phage vB_AhM_7 and PhAER-5 two genome sequences (as of June 30, 2025);

[0017] Figure 4 is the morphology diagram of the phage of the present application observed by transmission electron microscope;

[0018] Figure 5 is the conventional dilution distribution diagram of the phage of the present application;

[0019] Figure 6 is the one-step growth curve diagram of the phage of the present application;

[0020] Figure 7 is the thermal stability result diagram of the phage of the present application;

[0021] Figure 8 is the pH stability result diagram of the phage of the present application;

[0022] Figure 9 is the chloroform sensitivity result diagram of the phage of the present application;

[0023] Figure 10 is the bactericidal effect result diagram of the phage of the present application in vitro;

[0024] Figure 11 is the inhibition effect result diagram of the phage of the present application on biofilm;

[0025] Figure 12 is the protection effect result diagram of the phage of the present application on zebrafish artificially infected with Aeromonas hydrophila.

[0026] Biological preservation instructions

[0027] The application provides an Aeromonas hydrophila phage vB_AhM_7 which is preserved in the Guangdong Microbial Culture Collection Center, has a preservation number of GDMCC No: 66525-B1, and is preserved on June 17, 2025, and is located at No. 59, Building 5, Guangzhou Martyrs' Courtyard, 100, Xianlie Road. DETAILED DESCRIPTION

[0028] The application provides an Aeromonas hydrophila phage vB_AhM_7 which is preserved in the Guangdong Microbial Culture Collection Center on June 17, 2025, and has a preservation number of GDMCC No: 66525-B1.

[0029] The application further provides application of the Aeromonas hydrophila phage vB_AhM_7 in inhibiting Aeromonas hydrophila.

[0030] The application further provides application of the Aeromonas hydrophila phage vB_AhM_7 in inhibiting a biofilm of Aeromonas hydrophila.

[0031] The application further provides application of the Aeromonas hydrophila phage vB_AhM_7 in preparing a medicine for preventing and treating diseases caused by Aeromonas hydrophila. In the application, the diseases include gastrointestinal inflammation, wound infection, necrotizing fasciitis, septicemia, tail rot disease, ulcer disease, furuncle disease, enteritis disease and gill rot disease.

[0032] The application further provides application of the Aeromonas hydrophila phage vB_AhM_7 in preparing aquatic feed for preventing and treating diseases caused by Aeromonas hydrophila. In the application, the diseases include septicemia, tail rot disease, ulcer disease, furuncle disease, enteritis disease and gill rot disease.

[0033] The technical solutions provided by the application will be described in detail below in combination with examples, but they should not be understood as limitations to the protection scope of the application.

[0034] Host bacteria: Aeromonas hydrophila ATCC 7966 is purchased from the American Type Culture Collection.

[0035] Isolation and purification of Aeromonas hydrophila bacteriophage

[0036] a. Add CaCl2 to the sewage sample and let it stand overnight. Centrifuge and pass the water sample filtrate through a 0.45 μm filter membrane.

[0037] b. Mix the water sample filtrate with the host bacteria in 2x LB liquid medium, centrifuge and pass the filtrate through a 0.22 μm filter membrane.

[0038] c. Gradient dilute the filtrate with SM buffer, mix with the host bacteria, add to LB semi-solid medium, mix well, pour onto the lower solid nutrient agar medium, and incubate for 6-8 h.

[0039] d. Pick a single plaque and centrifuge at 8000 r / min for 10 min at 4°C, and pass through a 0.22 μm filter membrane.

[0040] e. Repeat c-d to obtain a single morphological plaque.

[0041] Experimental results: As shown in Figure 1 , the bacteriophage can form a transparent plaque on the plate with a diameter of about 1.5 mm, uniform in size and clear in edge.

[0042] f. Mix the bacteriophage with 50% glycerol at a ratio of 1:1 and store at -80°C.

[0043] Example 2 Isolation and sequence analysis of bacteriophage genome

[0044] (1) Isolation of bacteriophage genome

[0045] a. Add DNase I, RNase A, NaCl and PEG-8000 to the bacteriophage filtrate, centrifuge at 10000 r / min for 10 min at 4°C, resuspend the precipitate with appropriate amount of SM buffer, and obtain concentrated bacteriophage.

[0046] b. Use UNIQ-10 column virus genome extraction kit to extract the genome of the bacteriophage.

[0047] c. Send the bacteriophage genome extraction solution to Beijing Nuoweziyuan Technology Co., Ltd. and use Illumina Novaseq platform for high-throughput sequencing.

[0048] (2) Sequence analysis of bacteriophage genome

[0049] Figure 2 ANI comparison average nucleic acid identity is 95.57% (as of June 30, 2025) (as shown in Table 1 below) according to the ICTV definition of a new species, the ANI value <95% is defined as a new species, therefore Aeromonas hydrophila bacteriophage vB_AhM_7 and bacteriophage PhAER-5 are the same species, but bacteriophage PhAER-5 most protein function is predicted as hypothetical protein, only 4 functional proteins are predicted, while Aeromonas hydrophila bacteriophage vB_AhM_7 predicts the function of most proteins, therefore Aeromonas hydrophila bacteriophage vB_AhM_7 has research significance. Figure 3 ANI comparison average nucleic acid identity is 95.57% (as of June 30, 2025) (as shown in Table 1 below) according to the ICTV definition of a new species, the ANI value <95% is defined as a new species, therefore Aeromonas hydrophila bacteriophage vB_AhM_7 and bacteriophage PhAER-5 are the same species, but bacteriophage PhAER-5 most protein function is predicted as hypothetical protein, only 4 functional proteins are predicted, while Aeromonas hydrophila bacteriophage vB_AhM_7 predicts the function of most proteins, therefore Aeromonas hydrophila bacteriophage vB_AhM_7 has research significance.

[0050] Example 3 Biological characteristics of Aeromonas hydrophila bacteriophage vB_AhM_7

[0051] (1) Morphological observation of bacteriophage

[0052] a. Drop 1 drop of bacteriophage concentrate on the copper mesh, and stand at room temperature for 10 min.

[0053] b. Drop 1 drop of 2% phosphotungstic acid negative staining solution, stand for 2-3 min, dry and observe by transmission electron microscope.

[0054] Experimental results: as shown in Table 2 below, the bacteriophage is a myovirus, the head diameter is about 60 nm, the tail length is about 100 nm, and the structure is a regular icosahedron. Figure 4

[0055] (2) Titer and routine dilution of bacteriophage

[0056] a. Dilute the bacteriophage gradient with SM buffer, and after incubation of each dilution with the host bacteria, add it to the LB semi-solid culture medium, pour it on the lower solid agar culture medium, and culture for 6-8 h.

[0057] b. Choose plates with 30-300 plaques for counting, and calculate the titer of bacteriophage according to the following formula:

[0058] Bacteriophage titer (PFU / mL) = plaque number x dilution factor x 5.

[0059] Experimental results: the titer of Aeromonas hydrophila bacteriophage vB_AhM_7 can reach 10 9 PFU / mL.

[0060] c. Add host bacteria solution to the LB semi-solid culture medium, and pour it on the lower solid agar culture medium.

[0061] d. Dilute the bacteriophage gradient with SM buffer, and drop the dilution on the above-mentioned solidified medium, and culture for 6-8 h to observe the lysis. ​

[0062] Experimental results: such as Figure 5 As shown, the optimal dilution of Aeromonas hydrophila phage vB_AhM_7 is 10. -5 .

[0063] (3) Optimal Multiple of Infection (MOI) of bacteriophages

[0064] a. The spread plate method is used to calculate the number of bacteria, while the double-layer plate method is used to calculate the number of bacteriophages.

[0065] b. Mix the bacteriophage with the host bacteria at infection multiples of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001, add the mixture to LB liquid medium, and incubate for 6-8 hours.

[0066] c. Centrifuge the culture medium at 8000 r / min for 10 min at 4℃, and then filter it through a 0.22 μm filter membrane to determine the potency.

[0067] Experimental results: As shown in Table 1, the optimal multiplicity of infection for Aeromonas hydrophila phage vB_AhM_7 was 0.01.

[0068] Table 1 Optimal Multiplicity of Infection for Bacteriophages

[0069]

[0070] (4) One-step growth curve of bacteriophage

[0071] a. Mix the bacteriophage with the logarithmic-phase host bacteria at an MOI of 0.01 and allow it to adsorb for 15 min.

[0072] b. Centrifuge at 8000 r / min for 10 min at 4℃, resuspend three times in 2 mL of LB liquid medium, and then incubate in 18 mL of LB liquid medium.

[0073] c. Collect samples every 5 minutes for the first 25 minutes, and every 25 minutes from 25 to 200 minutes, and immediately determine the phage titer.

[0074] Experimental results: such as Figure 6 As shown, the incubation period of Aeromonas hydrophila phage vB_AhM_7 is about 15 min, the lysis period is 15-125 min, during which the number of phages increases rapidly, and the plateau period is 125-200 min, during which the number of phages is basically stable. The total lysis cycle is about 200 min. The lysis amount is calculated according to the following formula: lysis amount = phage titer at the end of the outbreak / host bacterial concentration at the beginning of infection.

[0075] The calculated lysis rate is approximately 272 PFU / cell.

[0076] (5) Thermostability and pH stability of the phage

[0077] a. The phage was placed in a 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C water bath, respectively, and sampled at 30 min and 60 min, and the titer was determined.

[0078] b. The pH of the LB liquid medium was adjusted to 1-13 with 2 mol / L NaOH and 1 mol / L HCl, and the titer was determined after mixing with the phage.

[0079] The experimental results are shown in Table 1 and Figure 1. Figure 7 and Figure 8 As shown in Table 1 and Figure 1, the Aeromonas hydrophila phage vB_AhM_7 still maintained a certain titer at 70°C, and had good high-temperature resistance; the titer of the Aeromonas hydrophila phage vB_AhM_7 was at a high level and stable in the range of pH 3-12, and could resist weak acid and weak base, and had good acid and alkali resistance.

[0080] (6) Chloroform sensitivity of the phage

[0081] The phage was mixed with chloroform at a ratio of 3:1, centrifuged at 10,000 r / min for 1 min at 4°C, and the titer was determined by taking the upper liquid, with no chloroform added as a blank control.

[0082] The experimental results are shown in Table 2 and Figure 2. Figure 9 As shown in Table 2 and Figure 2, the Aeromonas hydrophila phage vB_AhM_7 was not sensitive to chloroform, indicating that the phage shell had no lipid envelope and the structure was stable, and could resist the destruction of organic solvents.

[0083] Example 4

[0084] Inhibition of host bacteria by the Aeromonas hydrophila phage vB_AhM_7

[0085] (1) In vitro bacteriostasis of the phage

[0086] a. 150 μL of LB liquid medium was added to a 96-well plate, 50 μL of phage and 50 μL of host bacteria were added to the experimental group at MOI, and 50 μL of LB liquid medium and 50 μL of host bacteria or phage were added to the control group, and incubated at 30°C.

[0087] The experimental results are shown in Table 3 and Figure 3. Figure 10 As shown in Table 3 and Figure 3, with the extension of time, the OD 595nm value of the bacterial group increased continuously, and the addition of phage at MOI had a significant inhibitory effect on the host bacteria, and the OD 595nm of the phage-added group tended to be stable after 8 hours of treatment.

[0088] (2) Inhibition of biofilm by the phage

[0089] a. Experimental group: logarithmic phase host bacteria and bacteriophage were mixed in equal volume. Control group: logarithmic phase host bacteria and LB liquid medium were mixed in equal volume. Then, the mixture was cultured.

[0090] b. At 12h, 24h, 36h, 48h, samples were taken, centrifuged, and the culture medium was removed. Then, the bacteria were washed with 0.85% physiological saline (v / v) for 5 times, and the washing liquid was discarded. Finally, the bacteria were dried.

[0091] c. The bacteria were dyed with 0.1% crystal violet for 20 min, washed with 0.85% physiological saline (v / v) for 3-5 times, and then 33% glacial acetic acid was added to dissolve the bacterial membrane.

[0092] d. After ultrasonic oscillation, the OD was measured by using a 96-well plate. 595nm .

[0093] Experimental results: As shown in Table 1, the inhibition rate of Aeromonas hydrophila bacteriophage vB_AhM_7 on the biofilm of host bacteria ranged from 36.0% to 68.6%, which indicated that the bacteriophage had good ability to inhibit the biofilm of host bacteria. Figure 11

[0094] (3) Protective effect of bacteriophage on zebrafish

[0095] a. Zebrafish were divided into 6 groups, 10 fish in each group, and 3 repeats.

[0096] b. The first group was the control group, and the zebrafish were soaked in PBS buffer for 30 min. The second group was the bacterial artificial infection group, and the zebrafish were immersed in water containing host bacteria (final concentration of 1×10 7 CFU / mL) for 30 min. The third, fourth, and fifth groups were bacteriophage treatment groups, and the zebrafish were immersed in water containing host bacteria (final concentration of 1×10 7 CFU / mL) for 30 min, and then immediately immersed in water containing bacteriophage with a final concentration of 1×10 5 PFU / mL (third group), 1×10 6 PFU / mL (fourth group), and 1×10 7 PFU / mL (fifth group) for 30 min. The sixth group was the bacteriophage control group, and the zebrafish were immersed in water containing bacteriophage with a final concentration of 1×10 7 PFU / mL for 30 min.

[0097] c. After soaking, the zebrafish were kept in different beakers and not fed, and observed for 7 days.

[0098] Experimental results: As shown in Table 2, the survival rate of zebrafish in the bacteriophage treatment groups was significantly higher than that in the bacterial artificial infection group and the bacteriophage control group, which indicated that the bacteriophage had good protective effect on zebrafish. Figure 12 ​As shown, the survival rate of the host bacteria attack group of zebrafish was only 60% on the first day, while the survival rate of the low concentration vB_AhM_7 treatment group of zebrafish was 90% on the fifth day, and the survival rate of the high concentration vB_AhM_7 treatment group of zebrafish was 100% within seven days, indicating that Aeromonas hydrophila phage vB_AhM_7 has a protective effect on zebrafish.

[0099] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. An Aeromonas hydrophila bacteriophage vB_AhM_7, characterized in that, It was preserved in Guangdong Provincial Microbial Culture Collection on June 17, 2025, and the preservation number is GDMCC No: 66525-B1.

2. The bacteriophage Aeromonas hydrophila phage vB_AhM_7 of claim 1 for use in inhibiting Aeromonas hydrophila.

3. The bacteriophage Aeromonas hydrophila phage vB_AhM_7 of claim 1 for use in inhibiting Aeromonas hydrophila biofilm.

4. The bacteriophage Aeromonas hydrophila phage vB_AhM_7 of claim 1 for use in the preparation of a medicament for preventing / treating diseases caused by Aeromonas hydrophila.

5. Use according to claim 4, characterized in that, The diseases include gastrointestinal inflammation, diarrhea, wound infection, necrotizing fasciitis, cellulitis, respiratory tract infection, eye infection, septicemia, tail rot disease, ulcer disease, arthritis, furunculosis, enteritis disease, gill rot disease.

6. The bacteriophage Aeromonas hydrophila phage vB_AhM_7 of claim 1 for use in the preparation of animal feed for preventing / treating diseases caused by Aeromonas hydrophila.

7. Use according to claim 6, characterized in that, The diseases include septicemia, tail rot disease, ulcer disease, furunculosis, enteritis disease, gill rot disease.

Images