Giant virulent aeromonas hydrophila phage and application thereof

Abstract

The invention discloses a giant strong aeromonas hydrophila bacteriophage and application thereof, and belongs to the technical field of microorganisms. According to the invention, aeromonas hydrophila is taken as host bacteria, the phage is enriched from a collected lake water sample, and is separated and purified by adopting a double-layer agar plate method, so that a strain of phage is successfully obtained, the strain of phage is named as aeromonas hydrophila phage Cgtyf, the preservation number is CCTCC NO: M 2026099, and the titer of the phage can reach 1.0 * 10 < 10 > PFU / mL; the genome size of the phage is about 237 kb, and the phage belongs to a giant phage. In an in-vitro bacteriostasis experiment, the bacteriophage Cgtyf shows a remarkable bactericidal effect and a relatively strong growth inhibition capability on host bacteria, and shows a relatively high temperature tolerance range in a temperature stability test. Therefore, the bacteriophage Cgtyf provided by the invention can be effectively used for preventing and controlling aeromonas hydrophila in aquatic products, has a potential application value as an antibacterial drug substitute, is beneficial to reducing or avoiding the use of antibacterial drugs in aquaculture, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a virus, and more particularly to a giant, highly virulent Aeromonas hydrophila bacteriophage and its applications. Background Technology

[0002] Aeromonas hydrophila ( Aeromonas hydrophila Aeromonas hydrophila, belonging to the genus Aeromonas in the family Aeromonaceae, is a Gram-negative opportunistic pathogen widely distributed in freshwater ecosystems, aquatic products, and food processing environments. It exhibits strong environmental adaptability, surviving under varying temperatures, pH levels, and salinity. Furthermore, it can enhance its tolerance to adverse environments and antibiotics by forming biofilms. This bacterium produces various virulence factors, including hemolysin, extracellular enzymes, and secretory systems, playing crucial roles in host infection and pathogenesis. As a significant pathogen in freshwater aquaculture systems, Aeromonas hydrophila can infect various aquatic animals, causing severe economic losses. It can also be transmitted to humans through water or aquatic products, causing diseases such as gastroenteritis and septicemia, posing a potential threat to public health. Currently, the control of Aeromonas hydrophila infection mainly relies on antibiotics; however, long-term irrational use has led to the rapid emergence of drug-resistant strains, significantly reducing the effectiveness of prevention and control. Therefore, there is an urgent need to develop safe, efficient, and environmentally friendly alternative strategies.

[0003] Bacteriophages, as viruses that can specifically infect and lyse bacteria, possess advantages such as strong host specificity, ability to act on drug-resistant bacteria and biofilms, and minimal environmental impact, and are considered an important alternative or supplementary means to antibiotics. In recent years, bacteriophages have received widespread attention in medicine, food safety, and aquaculture, and research on bacteriophages targeting Aeromonas hydrophila has also been gradually carried out. CN120866239A discloses an Aeromonas hydrophila bacteriophage M01, which has an incubation period of approximately 10-30 minutes, an outbreak time of approximately 20-100 minutes, an outbreak dose of approximately 100-300 PFU / cell, and a maximum tolerated temperature of 50℃. CN120905162A discloses an Aeromonas hydrophila bacteriophage vB_AhM_7 with a genome size of 45.8 kb, an optimal multiplicity of infection (MOI) of 0.01, and a phage titer of 1×10⁻⁶ under optimal conditions. 9 The phage exhibits some tolerance to high temperatures, reaching up to 70 °C. However, after continuous treatment at high temperatures for 60 min, its potency significantly decreased, indicating that its thermal stability still has certain limitations.

[0004] The temperature tolerance of bacteriophages directly affects their stability during large-scale production, transportation, and storage, as well as their control efficacy in practical applications. However, currently screened Aeromonas hydrophila phages generally suffer from a narrow temperature tolerance range, limiting their application potential under complex environmental conditions. Therefore, it is necessary to further screen Aeromonas hydrophila phages with a wider temperature tolerance range and greater environmental adaptability from diverse ecological environments to enrich the existing phage resource library and provide a more solid resource foundation and theoretical support for the development of phage preparations and their application in aquatic disease control. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the first objective of the present invention is to provide a novel virulent Aeromonas hydrophila phage, which has a strong lytic ability against multiple strains of Aeromonas hydrophila and can be used for the prevention and control of Aeromonas hydrophila in the breeding environment or in animals.

[0006] To achieve the above-mentioned technical objectives, the inventors collected samples from Nanhu Lake, Yezhihu Lake, and sewage discharge areas in Wuhan City, Hubei Province, using Aeromonas hydrophila (… Aeromonas hydrophila The host bacteria were enriched with bacteriophages, and then isolated and purified using the double-layer agar plate method. A bacteriophage isolate with strong lytic activity against *Aeromonas hydrophila* was obtained. Subsequent purification yielded a bacteriophage monomer, identified as *Aeromonas hydrophila* bacteriophage, Latin name: Aeromonas hydrophila The phage, named Cgtyf, was deposited at the China Center for Type Culture Collection (CCTCC) on January 14, 2026, with accession number CCTCC NO: M 2026099. Based on observations of the phage and the morphology of its plaques, combined with whole-genome sequencing and comparative genomics analysis, it was identified as a novel giant, highly virulent Aeromonas hydrophila phage.

[0007] The Aeromonas hydrophila phage Cgtyf screened in this invention exhibits a typical long-tailed phage morphology, with an icosahedral head approximately 48 nm in diameter, a tail approximately 210 nm long, and a tail approximately 9.5 nm wide. Whole-genome sequencing results show that the genome length of Aeromonas hydrophila phage Cgtyf is 237,469 bp, classifying it as a giant phage. Comparative analysis reveals that this phage has an average nucleotide identity (ANI) of only 30% with currently reported phages, indicating its high phylogenetic uniqueness and potentially representing a new phage family.

[0008] Furthermore, under the optimal multiplicity of infection (MOI) of 0.1, the latency period of the phage Cgtyf of this invention is approximately 30 min, and the lysis period is approximately 80 min; during the stationary phase, the titer of phage Cgtyf can reach 1.0 × 10⁻⁶. 9 The lysis rate is approximately 212 PFU / cell, with a PFU / mL capacity.

[0009] The phage Cgtyf of this invention maintains a high titer and good stability after incubation at below 60 °C for 1 h, with no significant change in phage titer, remaining at 1.0 × 10⁻⁶. 8 The phage titer was above PFU / mL. When the treatment temperature was increased to 70 °C, the phage titer decreased by about two orders of magnitude; and after treatment at 80 °C for 1 h, no active phages were detected.

[0010] The bacteriophage Cgtyf of this invention maintains high activity after incubation at pH 5.0–10.0, with a titer maintained at 1.0 × 10⁻⁶. 9 Above PFU / mL. At pH 4.0, the phage titer decreased by approximately one order of magnitude, but remained above 1.0 × 10⁻⁶. 8 PFU / mL. In contrast, at pH 12.0, phage activity decreased significantly, retaining only about 68% of its activity; while under extremely acidic conditions (pH 2.0), the phage was completely inactivated.

[0011] The inhibitory effect of Aeromonas hydrophila phage Cgtyf on host bacteria was determined by measuring its effect. Within 72 hours of continuous treatment, the host bacterial concentration remained at a low level, indicating that the phage has a sustained and significant inhibitory effect on the host bacteria. Using the double-layer agar plate method, the host range of phage Cgtyf was determined with 12 strains of Aeromonas hydrophila as test subjects. The results showed that it could lyse 6 strains of Aeromonas hydrophila, with a lysis rate of 50%. These results indicate that phage Cgtyf has a broad lysis spectrum and good application potential. Therefore, the second objective of this invention is to provide the aforementioned Aeromonas hydrophila phage (… Aeromonas hydrophila The following applications of phage)Cgtyf are: (1) in the inhibition of Aeromonas hydrophila; (2) in the preparation of products for the prevention and / or treatment of diseases caused by Aeromonas hydrophila.

[0012] More preferably, the disease caused by Aeromonas hydrophila includes systemic bacterial septicemia in fish.

[0013] More preferably, the product is a drug or feed additive.

[0014] A third objective of this invention is to provide a product for preventing and treating Aeromonas hydrophila, wherein the active ingredient of the product comprises the aforementioned Aeromonas hydrophila bacteriophage (…). Aeromonas hydrophila phage)Cgtyf. More preferably, the dosage form of the product is a liquid preparation, a lyophilized preparation, or an oral solid preparation.

[0015] Compared with the prior art, the Aeromonas hydrophila phage Cgtyf provided by the present invention has the following beneficial effects: (1) The genome length of Aeromonas hydrophila phage Cgtyf of this invention is 237469 bp, belonging to giant phages. It is generally believed that giant phages with a genome size exceeding 200 kb usually carry more functional genes and have the potential to fight bacterial defense systems, which is of great value in phage evolution and application research. The results of mean nucleotide identity (ANI) analysis show that the highest ANI value of Aeromonas hydrophila phage Cgtyf of this invention compared with the reported phage genome is only 30.3%, which is significantly lower than the virologic family classification threshold recommended by ICTV (about 70%), indicating that this phage has high phylogenetic uniqueness.

[0016] (2) The highly virulent Aeromonas hydrophila phage Cgtyf of the present invention exhibits strong lytic activity against multiple strains of Aeromonas hydrophila, and can be effectively used for the control of Aeromonas hydrophila in aquaculture environments or animals. Its optimal multiplicity of infection is 10, and its titer can reach 1.0 × 10⁻⁶. 10 The bacteriophage exhibits a concentration of PFU / mL and maintains high activity at 30–70 °C, demonstrating excellent heat resistance. It also exhibits strong stability within a pH range of 3–12, showing good acid and alkali tolerance. Furthermore, it is insensitive to chloroform, structurally stable, and resistant to degradation by organic solvents. In in vitro experiments, it demonstrates significant bactericidal and inhibitory effects on host bacteria. Therefore, the bacteriophage provided by this invention can be used for the prevention and control of Aeromonas hydrophila in aquatic products, helping to avoid or reduce the use of antimicrobial drugs in aquaculture, and possesses significant practical application value and broad application prospects. Attached Figure Description

[0017] Figure 1 This is a transmission electron microscope schematic diagram of the Aeromonas hydrophila phage Cgtyf of the present invention; Figure 2 This is a genome analysis diagram of the Aeromonas hydrophila phage Cgtyf of this invention; Figure 3 This is the optimal MOI diagram for the Aeromonas hydrophila phage Cgtyf of this invention; Figure 4 This is a one-step growth curve of Aeromonas hydrophila phage Cgtyf according to the present invention; Figure 5 This is a graph showing the thermal stability results of the Aeromonas hydrophila phage Cgtyf of this invention; Figure 6 This is a graph showing the pH stability results of the Aeromonas hydrophila phage Cgtyf of this invention; Figure 7 This is a graph showing the in vitro bactericidal effect of the Aeromonas hydrophila phage Cgtyf of this invention. Detailed Implementation

[0018] This invention describes the screening and purification of Aeromonas hydrophila bacteriophages, followed by systematic identification and characterization of the resulting bacteriophage Cgtyf. This included morphological observation, host range determination, thermostability and pH stability analysis, determination of the optimal multiple of infection (MOI), one-step growth curve determination, and whole-genome sequencing analysis. The results showed that bacteriophage Cgtyf maintained high activity within the temperature range of 30–70 °C, exhibiting good thermostability; it also demonstrated strong stability under pH conditions of 3–12, showing good acid and alkali tolerance. In in vitro antibacterial experiments, bacteriophage Cgtyf exhibited significant bactericidal effects and strong growth inhibition capabilities against the host bacteria.

[0019] 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.

[0020] Example 1: Screening and purification of Aeromonas hydrophila bacteriophage The environmental samples used in this invention were collected in March 2024 from Nanhu Lake, Yezhihu Lake, and the sewage discharge area in Wuhan City, Hubei Province, including water and soil samples. The water samples were used directly for subsequent treatment, while the soil samples were filtered through gauze before use.

[0021] The phage enrichment procedure is as follows: Take 10 mL of water sample or soil filtrate, centrifuge at 10,000 r / min for 10 min, collect the supernatant and filter it through a 0.45 μm microporous membrane to obtain the water sample filtrate. Add 300 mL of the filtrate to LB liquid medium containing 3 times the nutrient concentration, and simultaneously inoculate with logarithmic-phase Aeromonas hydrophila as the host bacterium, and culture at 28 ℃ with shaking for 4–6 h. After the culture is completed, centrifuge at 5,000 × g for 10 min, collect the supernatant, and obtain the proliferated phage stock solution.

[0022] The isolation and purification of bacteriophages were performed using the double-layer agar plate method. 200 μL of the bacteriophage stock solution was mixed with 500 μL of Aeromonas hydrophila Ah-0 suspension, allowed to stand at room temperature for 10 min for adsorption, then mixed with 8 mL of LB semi-solid medium and quickly poured onto the surface of an LB solid medium plate. After solidification, the plate was inverted and incubated at 28 ℃ for 12–24 h until clear phage plaques appeared.

[0023] Single phage plaques exhibiting significant differences in morphology and size were picked using an inoculation loop, and the purification process was repeated three times to obtain single phage clones. The purified phages were then mixed with glycerol at a volume ratio of 8:2 and stored at −80 °C for later use.

[0024] Example 2: Morphological identification of bacteriophages

[0025] The purified phage sample from Example 1 was observed using a transmission electron microscope. The specific steps were as follows: the phage was immobilized on a 200-mesh copper grid coated with a 2–10 nm carbon film, and approximately 5 μL of phage suspension (1.0 × 10⁻⁶) was added. 10 (PFU / mL), allow to stand for 10 min for adsorption. After absorbing excess liquid with filter paper, dry on fresh filter paper for 1 min. Then, negatively stain the sample with 2% uranium acetate for 90 s, remove excess stain, gently rinse the copper mesh with ultrapure water, and dry on filter paper for 3 h.

[0026] The observations were performed using a transmission electron microscope (TEM) manufactured by Hitachi High Technology Co., Ltd., Japan, at an accelerating voltage of 80 kV. The TEM results are as follows: Figure 1 As shown, bacteriophage Cgtyf has a typical long-tailed bacteriophage morphology, with its head having an icosahedral structure, a diameter of approximately 48 nm, a tail length of approximately 210 nm, and a width of approximately 9.5 nm.

[0027] Example 3: Phage whole genome sequencing

[0028] After further amplification of the phage Cgtyf obtained in Example 1, 10 mL of high-titer phage suspension (1.0 × 10⁻⁶) was taken. 10 PFU / mL was used for genomic DNA extraction. Phage genomic DNA was extracted using the proteinase K / SDS method.

[0029] The specific steps are as follows: Take 750 μL of purified and filtered phage suspension, add DNase I and RNase A (both with a final concentration of 1 mg / L), and incubate at 37 ℃ for 15 min to remove free host nucleic acid. Then, treat at 80 ℃ for 30 min to fully inactivate the enzymes. Add EDTA (final concentration 0.02 mol / L), proteinase K (final concentration 50 mg / mL), and lysis buffer containing 0.5% SDS sequentially, and lyse at 56 ℃ for 1.5 h. After lysis, add an equal volume of equilibrated phenol for extraction, centrifuge at 12,500 r / min for 15 min, and transfer the upper aqueous phase to a new centrifuge tube; then add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), mix gently, and centrifuge at 12,000 r / min for 15 min to further remove protein and polysaccharide impurities. After collecting the upper aqueous phase, an equal volume of isopropanol was added, and the mixture was allowed to stand at 20 °C for 5 h. The DNA precipitate was then collected by centrifugation at 12,500 r / min for 20 min. The precipitate was washed with pre-cooled 75% ethanol, dried at room temperature, and resuspended in 30 μL of deionized water to obtain phage genomic DNA.

[0030] The obtained DNA was used for whole-genome sequencing using the Illumina NextSeq sequencing platform. The phage genome diagram is shown below. Figure 2 As shown in the figure. The results showed that the complete genome of bacteriophage Cgtyf is 237469 bp in length, belonging to giant bacteriophages. Its genome is double-stranded DNA (dsDNA) with a GC content of 44.3%. BLAST alignment analysis did not find any sequences with high homology to previously reported bacteriophages. Functional annotation using the RAST platform predicted a total of 241 open reading frames (ORFs), of which 46 encode known functional proteins and the remaining 195 are putative proteins. In addition, 4 transfer RNAs (tRNAs) were also detected.

[0031] Example 4: Host profile determination of bacteriophage Cgtyf

[0032] The host range of bacteriophage Cgtyf was determined using the standard drop method. A total of 12 Aeromonas hydrophila isolates were selected as test subjects (see Table 1). The specific method was as follows: each strain was cultured on a double-layer agar plate to the logarithmic growth phase, and then 10 μL of high-titer bacteriophage suspension (1.0 × 10⁻⁶) was taken. 8 PFU / mL was added to the surface of the plate, and after the droplet was naturally adsorbed, it was incubated overnight at 28 °C and the formation of clear lysis spots was observed.

[0033] The results are shown in Table 1. Among the 12 Aeromonas hydrophila strains tested, bacteriophage Cgtyf was able to lyse 6 of them, with a lysis rate of 50%, indicating that the bacteriophage has a relatively wide host range.

[0034] Table 1. Host spectrum of bacteriophage Cgtyf

[0035]

[0036] Example 5: Optimal Multiplicity of Infection for Bacteriophage Cgtyf

[0037] The host bacteria were cultured to the logarithmic growth phase. A suspension of the host bacteria in the logarithmic growth phase was then taken, and phage stock solutions with multiplicity of infection (MOI) of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 were added, respectively. The mixtures were incubated in a constant temperature shaking incubator at 28 ℃ and 180 r / min for 8 h. After incubation, the mixtures were centrifuged at 5000 × g for 10 min, and the supernatant was collected. The phage titers of each treatment group were determined using the double-layer agar plate method, and the MOI corresponding to the highest phage titer was taken as the optimal MOI. The results are as follows: Figure 3 As shown, the titer of phage Cgtyf was highest when the MOI was 10, reaching 2.25 × 10⁻⁶. 10 The PFU / mL level was used to determine the optimal multiplicity of infection (MWI) as 10.

[0038] Example 6: One-step growth curve of bacteriophage Cgtyf

[0039] At the optimal multiplicity of infection (MOI) of 10, the bacteriophage was mixed with the host bacteria and incubated in a 28 °C water bath for 20 min to allow the bacteriophage to fully adsorb onto the surface of the host bacteria. Subsequently, the mixture was centrifuged at 12000 × g for 5 min, and the supernatant was discarded. The bacterial pellet was resuspended in 1 mL of LB liquid medium, centrifuged again, and the supernatant was discarded. This washing step was repeated twice to remove any unadsorbed free bacteriophage.

[0040] The precipitate was resuspended in 5 mL of LB liquid medium and incubated in a constant temperature shaking incubator at 28 ℃ and 180 r / min, with timing started simultaneously. A 100 μL sample was taken every 10 min until the experiment ended after 3 h. The phage titer at each time point was determined using the double-layer agar plate method, and a one-step growth curve was plotted with infection time on the x-axis and phage titer on the y-axis. Results are as follows: Figure 4As shown, under an MOI of 10, the latency period of phage Cgtyf is approximately 30 min, and the lysis period is approximately 80 min. During the stationary phase, the phage titer can reach 1.0 × 10⁻⁶. 8 The lysis rate is approximately 212 PFU / cell, with a PFU / mL capacity.

[0041] Example 7: Thermal stability analysis of bacteriophage Cgtyf

[0042] Equal volumes of phage stock solution were aliquoted into 1.5 mL EP tubes and incubated in water baths at 4 ℃, 20 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, and 80 ℃ for 1 h. Immediately after incubation, the samples were removed, and the phage titer was determined using the double-layer agar plate method. Results are as follows: Figure 5 As shown, phage Cgtyf maintained a high titer and good stability after incubation at below 60 °C for 1 h, with no significant change in phage titer, remaining at 1.0 × 10⁻⁶. 8 The concentration of phages was above PFU / mL. When the treatment temperature was increased to 70 °C, the phage titer decreased by about two orders of magnitude; however, after treatment at 80 °C for 1 h, no active phages were detected, indicating that high temperature can significantly affect their activity.

[0043] Example 8: pH stability analysis of bacteriophage Cgtyf

[0044] To assess the stability of bacteriophage Cgtyf under different pH conditions, bacteriophage suspensions were placed in buffer solutions with pH values ​​ranging from 2.0 to 12.0 and incubated at room temperature for 1 h. After incubation, the titers of bacteriophages in each treatment group were determined using the double-layer agar plate method. The results are as follows: Figure 6 As shown, bacteriophage Cgtyf maintained high activity after incubation at pH 5.0–10.0, with the titer remaining at 1.0 × 10⁻⁶. 9 Above PFU / mL. At pH 4.0, the phage titer decreased by approximately one order of magnitude, but remained above 1.0 × 10⁻⁶. 8 PFU / mL. In contrast, at pH 12.0, phage activity decreased significantly, retaining only about 68% of its activity; while under extremely acidic conditions (pH 2.0), the phage was completely inactivated, indicating that its capsid structure underwent irreversible denaturation under these conditions.

[0045] Example 9: Inhibitory effect of bacteriophage Cgtyf on Aeromonas hydrophila proliferation in broth medium

[0046] The phage suspension was serially diluted to obtain different multiples of infection (MOIs) of 10, 1, and 10⁻⁶. -1 and 10 -2 In a 96-well microplate, 100 μL of phage suspension corresponding to the MOI was added to each well, mixed with 100 μL of host bacterial culture in the exponential growth phase. Wells containing 100 μL of LB medium and 100 μL of SM buffer served as blank controls.

[0047] The microplates were placed in an automated microbial growth curve analyzer and incubated at 28 °C. The optical density (OD) at 600 nm was measured every 30 min for 24 h or 72 h. 600 (With incubation time as the x-axis, OD) 600 The values ​​are plotted on the ordinate to show bacterial growth and lysis curves, in order to analyze the inhibitory dynamics of bacteriophages on host bacteria under different MOI conditions.

[0048] The results are as follows Figure 7 As shown, bacteriophage Cgtyf exhibited a significant inhibitory effect on Aeromonas hydrophila, continuously inhibiting the growth of the host bacteria for 72 hours. The inhibitory effect was most significant when the MOI was 10: host bacterial growth was slow from 0 to 22 hours, the bacterial count decreased significantly from 22 to 42 hours, and the OD value increased significantly from 42 to 72 hours. 600 The MOI value remained stable at approximately 0.7, and the number of host bacteria was consistently significantly lower than that of the control group throughout the 72-hour incubation period. Overall, the inhibitory effect of the phage on the host bacteria gradually increased with the increase of the MOI value, exhibiting a good dose-dependent effect.

Claims

1. A strain of Aeromonas hydrophila bacteriophage ( Aeromonas hydrophila phage)Cgtyf, its accession number is CCTCC NO: M 2026099.

2. The Aeromonas hydrophila phage as described in claim 1 ( Aeromonas hydrophila Application of phage)Cgtyf in inhibiting Aeromonas hydrophila.

3. The Aeromonas hydrophila phage as described in claim 1 ( Aeromonas hydrophila The use of phage)Cgtyf in the preparation of products for the prevention and / or treatment of diseases caused by Aeromonas hydrophila.

4. The application according to claim 3, characterized in that, The diseases caused by Aeromonas hydrophila include systemic bacterial septicemia in fish.

5. The application according to claim 3, characterized in that, The product in question is a drug or feed additive.

6. A product for preventing and controlling Aeromonas hydrophila, characterized in that, The active ingredient of the product contains the Aeromonas hydrophila phage as described in claim 1. Aeromonas hydrophila phage)Cgtyf.

7. The product according to claim 6, characterized in that, The product is available in liquid, lyophilized, or oral solid dosage forms.

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