Aeromonas hydrophila bacteriophage vBAhSLZ and application thereof

Abstract

The invention discloses an aeromonas hydrophila bacteriophage vBAhSLZ and application thereof, and belongs to the technical field of microorganisms. According to the invention, aeromonas hydrophila is taken as host bacteria, a bacteriophage vBAhSLZ is separated from a sewage sample collected from a sewage outlet of a holothurian culture base, and the titer of the bacteriophage is 109 PFU / mL; when the temperature is 70 DEG C or below and the pH value is 3-10, the activity is relatively high; the phage is not sensitive to chloroform, and a lipid envelope does not exist outside a phage capsid, so that the structure of the phage is not damaged by chloroform; the in-vitro sterilization capability on host bacteria is relatively high, and the highest inhibition rate on a biofilm can reach about 60%; in a food matrix, the growth of aeromonas hydrophila in salmons can be effectively inhibited; aeromonas hydrophila in aquatic products can be effectively controlled, the use of antibacterial agents is reduced, and the method has huge application potential in food preservation and biological prevention and control.

Description

Technical Field

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

[0002] Aeromonas hydrophila is a common aquatic pathogen widely distributed in various environments, including freshwater, seawater, and soil. This bacterium is not only one of the most serious pathogens in aquaculture, causing septicemia, enteritis, and skin ulcers in freshwater fish, shrimp, crabs, and other aquatic animals, resulting in significant economic losses; it is also an important zoonotic pathogen, capable of infecting humans through contaminated water or food, leading to gastroenteritis, wound infections, and even septicemia.

[0003] Currently, the prevention and control of Aeromonas hydrophila mainly rely on antibiotics and chemical disinfectants. However, the long-term, widespread, and unregulated use of these drugs has led to a series of serious problems: ① Increasingly prominent bacterial resistance: The overuse of antibiotics has led to the emergence and spread of multidrug-resistant and even pan-drug-resistant Aeromonas hydrophila strains, significantly reducing or even completely eliminating the efficacy of many traditional antibiotics, posing a great challenge to clinical treatment and aquatic disease control. ② Drug residues and food safety issues: The use of antibiotics and chemical drugs in aquaculture may result in drug residues in aquatic products, which can then enter the human body through the food chain, threatening human health and affecting the export and trade of aquatic products. ③ Environmental pollution and ecological damage: Unmetabolized antibiotics and chemical disinfectants are released into the natural environment, disrupting the microecological balance of aquatic bodies, causing toxic effects on non-target organisms, and promoting the spread of drug-resistant genes in the environment. Therefore, the development of a new type of antibacterial agent that can effectively replace or supplement antibiotics and has high efficiency, safety, and environmental protection characteristics is urgently needed.

[0004] Bacteriophages, or viruses that infect bacteria, are the most abundant biological entities in nature. They can specifically infect and lyse host bacteria and possess the ability to self-replicate. Based on these characteristics, bacteriophage biocontrol technology shows great application potential: ① High specificity: They typically target only specific pathogens without disrupting the normal microbial community in the environment. ② Self-replication: They can rapidly multiply in the presence of host bacteria, providing long-lasting efficacy. ③ Environmentally friendly: They leave no chemical residues and can be degraded by microorganisms in the natural environment. ④ Overcoming drug resistance: Their bactericidal mechanism is completely different from that of antibiotics, effectively lysing drug-resistant strains.

[0005] There is a lack of a specific anti-Aeromonas hydrophila method in the existing technology that can effectively overcome bacterial resistance, avoid drug residues, and is ecologically safe. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a bacteriophage that can efficiently lyse Aeromonas hydrophila and its application.

[0007] The technical solution of the present invention is as follows: an Aeromonas hydrophila phage vB_AhS_LZ, deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67232-B1, deposited on November 6, 2025, and deposited at the address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou.

[0008] Biological agents containing the aforementioned Aeromonas hydrophila phage vB_AhS_LZ.

[0009] The aforementioned Aeromonas hydrophila bacteriophage vB_AhS_LZ or biological agents are used in the preparation of drugs for the prevention or treatment of diseases caused by Aeromonas hydrophila, such as gastroenteritis, diarrhea, wound infections, necrotizing fasciitis, cellulitis, respiratory infections, eye infections, sepsis, tail rot, ulcer disease, arthritis, furuncles, enteritis, or gill rot caused by Aeromonas hydrophila.

[0010] The aforementioned Aeromonas hydrophila bacteriophage vB_AhS_LZ or biological agents are used for the prevention and control of Aeromonas hydrophila for non-disease treatment purposes, such as food preservation, water sterilization, and feed antibacterial activity.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] This invention uses Aeromonas hydrophila as the host bacterium to isolate a bacteriophage vB_AhS_LZ from wastewater samples collected from the discharge outlet of a sea cucumber farming base. The titer of this bacteriophage is 10. 9 PFU / mL; exhibits high activity below 70 ℃ and at pH 3~10; insensitive to chloroform, and lacking a lipid envelope on its capsid, its structure is not disrupted by chloroform; possesses strong bactericidal ability against host bacteria in vitro, with an inhibition rate of up to approximately 60% on biofilms; effectively inhibits the growth of Aeromonas hydrophila in grass carp in food processing; effectively controls Aeromonas hydrophila in aquatic products, reducing the use of antibiotics, and has great application potential in food preservation and biocontrol.

[0013] Preservation information:

[0014] The Aeromonas hydrophila phage vB_AhS_LZ provided by this invention is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67232-B1, deposit date November 6, 2025, and deposit address 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0015] Figure 1 Morphage vB_AhS_LZ morphology diagram;

[0016] Figure 2 Transmission electron microscopy image of bacteriophage vB_AhS_LZ;

[0017] Figure 3 Image for identifying the vB_AhS_LZ phage gene type;

[0018] Figure 4 This is a one-step growth curve of bacteriophage vB_AhS_LZ;

[0019] Figure 5 Figure (b) shows the experimental results of thermal stability (a) and pH stability of bacteriophage vB_AhS_LZ;

[0020] Figure 6 Figure showing the chloroform susceptibility test results of bacteriophage vB_AhS_LZ;

[0021] Figure 7 The image shows the results of the in vitro bactericidal experiment of bacteriophage vB_AhS_LZ;

[0022] Figure 8 The graph shows the inhibitory effect of bacteriophage vB_AhS_LZ on biofilms.

[0023] Figure 9 The graph shows the inhibitory effect of bacteriophage vB_AhS_LZ on Aeromonas hydrophila in salmon.

[0024] Preservation Information

[0025] Aeromonas hydrophila phage vB_AhS_LZ is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67232-B1, deposited on November 6, 2025, at the address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Detailed Implementation

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0027] Example 1: Isolation, purification, and identification of bacteriophages

[0028] Wastewater was collected from the sewage outlet of a sea cucumber farming base in Linghai City, Liaoning Province. Bacteriophages were separated and purified using the double-layer agar plate method.

[0029] Separation: CaCl2 was added to the collected wastewater samples, and the mixture was allowed to stand overnight. After centrifugation at 8000 r / min for 10 min, the samples were filtered through a 0.45 μm filter membrane. The filtrate was added to an equal volume of 2×LB liquid medium, and Aeromonas hydrophila bacterial suspension was added and mixed well. The mixture was then incubated overnight at 30 ℃ with shaking. The next day, the supernatant was centrifuged at 8000 r / min for 10 min and filtered through a 0.22 μm filter membrane. The filtrate was serially diluted with SM buffer, mixed with the host bacteria, and then added to LB semi-solid medium. The mixture was poured onto the lower agar medium and incubated upside down for 8 h. Plaques were observed.

[0030] Purification: Select clear, single phage plaques and incubate them in SM buffer at 30 °C for 6-8 hours on a shaker. Centrifuge at 8000 rpm for 10 minutes, collect the supernatant, and filter it through a 0.22 μm filter membrane. Dilute the filtrate as described above and pour it into a double-layer agar plate. Repeat this step 3-5 times to obtain phage plaques of uniform size and clear appearance. Figure 1 ).

[0031] The purified phage titer can reach 10. 9 PFU / mL, phage fluid mixed with an equal volume of 50% glycerol and stored at -80°C.

[0032] Phage genome sequencing and analysis

[0033] DNase I, RNase A, NaCl, and PEG-8000 were added to the phage filtrate, and the mixture was centrifuged at 10,000 r / min for 10 min at 4 ℃. The precipitate was resuspended in an appropriate amount of SM buffer to obtain concentrated phage. The phage genome was extracted using the UNIQ-10 column-based viral genome extraction kit. The extracted phage genome was sent to Beijing Novogene Technology Co., Ltd. for high-throughput sequencing using the Illumina Novaseq next-generation sequencing platform.

[0034] The genome of Aeromonas phage vB_AhS_LZ contains 45 open reading frames and belongs to the family Siphoviridae. It lacks tRNA. The VFDB database was used to predict whether the phage carries virulence genes, and the ResFinder online tool was used to predict whether it carries drug resistance genes. The results showed that neither virulence nor drug resistance genes were detected.

[0035] Phylogenetic tree constructed by BLASTing the genome in GenBank revealed that it is in the same branch as Escherichia coli phage vB_EcoP_S523 (YP 009802477.1), indicating that it is most closely related and has a high identity value (94%) with phage vB_EcoP_S523 (as of October 17, 2025).

[0036] The bacteriophage was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 6, 2025.

[0037] Morphage morphology observation:

[0038] Negative staining was used to stain the bacteriophages: 10 μL of concentrated bacteriophage suspension was dropped onto a copper grid, allowed to dry at room temperature, and then stained with 2% phosphotungstic acid. The morphology was observed under a transmission electron microscope (80 kV). At 500.0 nm, the bacteriophage was observed to have an icosahedral head, approximately 80 nm in diameter, and a tail sheath approximately 100 nm long, classifying it as a long-tailed bacteriophage. Figure 2 ).

[0039] Phage genotype identification:

[0040] The bacteriophage genome was treated with DNase I, RNase A, and mung bean nuclease, and the results showed that ( Figure 3 It can be completely degraded by DNase I, but not by RNase A and mung bean nuclease, indicating that the genome is a double-stranded DNA structure.

[0041] Example 2: Biological characteristics of bacteriophage vB_AhS_LZ

[0042] (1) Optimal multiple of infection (MOI) for bacteriophages:

[0043] The concentration of bacterial suspension in the logarithmic phase was determined using the viable cell count method. The bacterial suspension was serially diluted 10-fold, and bacteriophages were mixed with *Aeromonas hydrophila* at multiplicity of infection (MOI) values ​​of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001, then added to LB broth and incubated for 6–8 h. The mixture was centrifuged at 8000 r / min for 10 min at 4 °C, and the supernatant was filtered through a 0.22 µm filter to obtain the bacteriophage fluid. The titer was determined using the double agar plate method. The highest titer, reaching 5 × 10⁻⁶, was observed at an MOI of 0.1.11 PFU / mL (Table 1).

[0044] Table 1. Optimal Multiplicity of Infection for Bacteriophages

[0045]

[0046] (2) One-step growth curve of bacteriophage:

[0047] The bacteriophage was mixed with logarithmic-phase Aeromonas hydrophila at an MOI of 0.1, adsorbed for 15 min, centrifuged at 8000 r / min for 5 min at 4 ℃, the supernatant was discarded, and the precipitate was resuspended in LB liquid medium. This step was repeated 3 times to remove free bacteriophage. After the last resuspension, all bacterial culture was transferred to LB liquid medium to a final volume of 20 mL. Samples were taken every 10 min, and the titer was determined immediately. The phage had a latency period of 15 min, a lysis period of 15-150 min, and then entered a stationary phase. Figure 4 ).

[0048] (3) Phage thermal stability and pH stability:

[0049] With an initial valence of 10 9 Phages of PFU / mL were aliquoted and treated at 0 ℃, 4 ℃, 20 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, and 80 ℃ for 60 min, respectively. Samples were taken every 30 min to determine the titer.

[0050] Adjust the pH of LB liquid medium to 2-13 with 1 mol / L NaOH and 1 mol / L HCl, then mix with bacteriophages to determine titer.

[0051] The bacteriophages are stable below 70 °C and essentially inactivated at 70 °C and above. They are stable at pH 3-10 and exhibit good acid and alkali tolerance. Figure 5 ).

[0052] (4) Phage chloroform susceptibility:

[0053] The phage fluid was mixed with chloroform at a ratio of 100:1, allowed to stand for 30 min, centrifuged at 10000 r / min for 1 min at 4 ℃, and the supernatant was collected to determine the titer. The phage without chloroform served as a blank control. The phage's insensitivity to chloroform indicates that the phage coat lacks a lipid membrane, thus resisting damage from organic solvents. Figure 6 ).

[0054] (5) In vitro bacteriophage bactericidal experiment:

[0055] Add 100 μL of Aeromonas hydrophila culture grown to the logarithmic phase to each well of a 96-well plate. Add equal volumes of phage solution at different dilutions to achieve MOIs of 1, 0.1, 0.01, and 0. Bracken LB medium is added to bring the total volume to 200 μL per well. For the control group (MOI 0), add 100 μL of bacterial culture + 100 μL of LB medium. Each row of wells constitutes one group. Incubate at 30 ℃. OD is measured every 1 h using a microplate reader. 595nm Value. Over time, the OD value of the control group... 595nm The value continued to increase, and the phage in the MOI group (0.1) showed the best inhibitory effect on the host bacteria, with the trend tending to stabilize. Figure 7 ).

[0056] (6) Inhibition of biofilms by bacteriophages:

[0057] Add 100 μL of Aeromonas hydrophila culture grown to the logarithmic phase to each well of a 96-well plate. Add an equal volume of bacteriophage fluid according to the optimal MOI. Wells containing only culture medium and only bacterial culture serve as blank and negative controls, respectively. Samples are taken at 12 h, 24 h, 36 h, and 48 h. The culture medium is removed, and the plates are washed with 0.85% sterile physiological saline until the solvent is clear. After removing any residual liquid, the plates are dried at room temperature for 30 min. Then, 200 μL of 1% crystal violet solution is added and discarded. The plates are then washed several times with sterile physiological saline until the solvent is clear. 200 μL of 33% glacial acetic acid is added to each well to break up the biofilm adhering to the wells. After slow shaking, the OD is measured. 595nm Value. The phage's inhibition rate against biofilm can reach about 60%, indicating that this phage has a good ability to inhibit host bacterial biofilm. Figure 8 ).

[0058] Example 3: Inhibition of Aeromonas hydrophila by bacteriophages in grass carp

[0059] Take 100 μL 1×10 6 Aeromonas hydrophila at cfu / mL was inoculated onto the surface of aseptically treated salmon samples and allowed to stand for 20 min to allow for adsorption. 100 μL of phages with different titers (corresponding to MOIs of 1, 0.1, 0.01, and 0) were added to the surface of grass carp artificially contaminated with Aeromonas hydrophila. The negative control group used SM buffer instead of phages. After storing the samples at 4 ℃ for a certain period, they were then plated onto RS plates. The results showed (…). Figure 9In a 4 ℃ cold chain environment (simulating salmon storage conditions), with the untreated group as the control, phage treatment groups with MOIs of 10, 1, 0.1, and 0.01 were set up. The changes in pathogen counts were monitored over 120 hours (5 days, covering the salmon shelf life). The results showed that all treatment groups had significant antibacterial effects, exhibiting a dose-response relationship. The MOI=10 group showed the most significant effect, reducing pathogen counts by approximately 4.7 log CFU / mL compared to the control, achieving strong clearance. The MOI=1 and MOI=0.1 groups showed similar effects, reducing pathogen counts by approximately 3.32 log CFU / mL and 4.36 log CFU / mL respectively, while still maintaining stable antibacterial activity, potentially reducing application costs. The MOI=0.01 group is presumably less effective than the previous groups, but better than the control. In summary, phages are effective against salmon pathogens at low temperatures, and the higher the MOI, the stronger the effect. This provides a reference for practical applications.

Claims

1. An Aeromonas hydrophila phage strain vB_AhS_LZ was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 6, 2025, with accession number GDMCC No: 67232-B1.

2. A biological agent containing Aeromonas hydrophila phage vB_AhS_LZ as described in claim 1.

3. The use of Aeromonas hydrophila phage vB_AhS_LZ as described in claim 1 or the biological agent as described in claim 2 in the preparation of a drug for the prevention or treatment of diseases caused by Aeromonas hydrophila.

4. The use of Aeromonas hydrophila phage vB_AhS_LZ as described in claim 1 or the biological agent as described in claim 2 in the prevention and control of Aeromonas hydrophila for non-disease treatment purposes.

Images