Vibrio alginolyticus bacteriophage vBValPR38Z with strong splitting property and application thereof

Abstract

The invention discloses a vibrio alginolyticus bacteriophage vBValPR38Z with a strong lysis property and an application of the vibrio alginolyticus bacteriophage The invention discloses a vibrio alginolyticus phage vBValPR38Z, and the preservation number of the vibrio alginolyticus phage vBValPR38Z is as follows: GDMCC (Graphical Digital Microorganism Center for Culture Collection Center) NO: 67439-B1. The bacteriophage keeps relatively high activity under the conditions that the temperature is 4-25 DEG C and the pH is 7-8. The bacteriophage R38Z has the characteristics of rapid adsorption and short incubation period, can significantly inhibit the growth of a host in the early stage of infection, shows efficient lysis ability, and can rapidly complete adsorption, replication and lysis processes. Genome analysis shows that the R38Z has high genetic safety, the genome of the R38Z is free of virulence, resistance and lyogen related genes, and meanwhile, the R38Z carries various tRNA genes, so that the protein translation efficiency and infection adaptability can be optimized. The characteristics jointly form important advantages of the R38Z in bacteriophage treatment application.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a highly lytic Vibrio alginolyticus bacteriophage vB_ValP_R38Z and its applications. Background Technology

[0002] With the continuous expansion of aquaculture scale and the improvement of intensification, problems such as eutrophication, ecological imbalance, and the spread of pathogenic microorganisms in the aquatic environment have become increasingly prominent. Among them, the frequent occurrence of bacterial diseases has become a major bottleneck restricting the sustainable development of the industry, not only leading to increased mortality and economic losses in farmed animals, but also threatening the ecological security of aquatic areas. Vibrio is an important pathogenic bacterium for many marine animals, widely distributed in marine, freshwater, and saline environments such as estuaries. Vibrio diseases caused by Vibrio are characterized by rapid spread, high pathogenicity, and high mortality, which can cause serious diseases and even large-scale deaths in aquatic animals such as fish, shrimp, and shellfish. Its typical pathogenic process includes: pathogen adhesion to the host surface and colonization, invasion of tissues, proliferation in the body, and production of toxins, ultimately leading to the death of the host. Among them, Vibrio alginolyticus is a common highly pathogenic bacterium in coastal aquaculture, with obvious regional epidemic characteristics, which can lead to significant morbidity and mortality, and cause serious economic losses to the aquaculture industry.

[0003] Currently, antibiotics remain the most commonly used measure for controlling vibriosis. However, antibiotic resistance and its risk of spread in aquatic environments have attracted widespread attention. Although non-antibiotic methods such as vaccines, probiotics, immunostimulants, and water conditioners can inhibit the occurrence of vibriosis to some extent, their control effects are often limited by long research and development cycles, slow action, and high environmental sensitivity. In view of this, phage therapy, as a highly specific, biodegradable, and environmentally friendly natural antibacterial strategy, is gradually becoming a hot topic in aquatic disease control research. Phage therapy is a two-step process, involving the phage penetrating the target bacteria and then killing them. A bacteriophage is a bacterial virus with no cellular structure, mainly composed of a protein capsid and nucleic acid containing genetic material. Phages can be divided into virulent phages and temperate phages. Virulent phages can invade bacterial cells, destroy the cell wall through enzymatic action, and utilize host resources to complete biosynthesis and assembly, causing bacterial lysis. Phage therapy is a promising alternative to antibiotics, but most phages suffer from slow adsorption and long latency periods. Therefore, it is imperative to find Vibrio alginolyticus phages with rapid lysis capabilities and genetic safety. Summary of the Invention

[0004] The purpose of this invention is to provide Vibrio alginolyticus phage vB_ValP_R38Z and its applications, providing technical support for the development of bio-agriculture and related industries.

[0005] The first objective of this invention is to provide Vibrio alginolyticus phage vB_ValP_R38Z, with accession number GDMCC NO: 67439-B1.

[0006] The second objective of this invention is to provide a microbial preparation for controlling Vibrio alginolyticus or Vibrio erwinii, which contains the aforementioned bacteriophage vB_ValP_R38Z as an active ingredient.

[0007] A third object of the present invention is to provide the use of the above-mentioned bacteriophage vB_ValP_R38Z or the above-mentioned microbial preparation in the prevention and / or killing of Vibrio alginolyticus or Vibrio erwinis.

[0008] A fourth object of the present invention is to provide the use of the above-mentioned bacteriophage vB_ValP_R38Z or the above-mentioned microbial preparation in the preparation of products for the prevention and / or killing of Vibrio alginolyticus or Vibrio erwinis.

[0009] Preferably, the Vibrio alginolyticus and Vibrio erwinii are derived from the water used for Litopenaeus vannamei farming.

[0010] Preferably, the Vibrio alginolyticus is Vibrio alginolyticus D69-TY10.

[0011] Preferably, the product includes pharmaceuticals, disinfectants, water purifiers, feed or feed additives.

[0012] A fifth object of the present invention is to provide a product for controlling Vibrio alginolyticus or Vibrio erwinii infection, which contains the above-mentioned bacteriophage vB_ValP_R38Z or the above-mentioned microbial preparation.

[0013] Preferably, the Vibrio alginolyticus is Vibrio alginolyticus D69-TY10.

[0014] The sixth objective of this invention is to provide a method for preventing harmful infections caused by Vibrio alginolyticus or Vibrio erwinii in vitro, which utilizes the lytic action of the aforementioned bacteriophage vB_ValP_R38Z or the aforementioned microbial preparation to kill Vibrio alginolyticus or Vibrio erwinii in vitro.

[0015] This invention systematically characterized a Vibrio alginolyticus phage, vB_ValP_R38Z, derived from aquaculture environment samples. Host spectrum testing showed that this phage could infect Vibrio alginolyticus and Vibrio Erwinis from aquaculture environments. Infection kinetics analysis revealed that vB_ValP_R38Z exhibited rapid adsorption and a short latency period, significantly inhibiting host growth in the early stages of infection and demonstrating highly efficient lytic activity. Genomic analysis showed that this phage lacked any toxic, resistance, or lysogenic genes. Furthermore, this phage carried multiple helper metabolic genes and contained three tRNA genes (Ile, Pro, and Ser), indicating a certain degree of translational system independence and metabolic regulation during infection. Environmental stability experiments showed that this phage maintained high activity at 4℃–25℃ and pH 7–8, making it suitable for the physicochemical conditions of aquaculture environments. In summary, vB_ValP_R38Z combines rapid lytic activity with genetic safety, demonstrating potential application as a safe and efficient Vibrio control factor in aquaculture systems.

[0016] This invention characterizes the biological properties and genomic features of a novel Vibrio shorttail bacteriophage, R38Z, isolated from wastewater from the Xiamen Xiashang Aquatic Products Market. R38Z can infect hosts originating from aquaculture environments and, in practical applications, can specifically recognize and lyse specific pathogens without disrupting the environmental microbial balance. Infection kinetics results show that this bacteriophage exhibits significant lysis efficiency, demonstrating strong antibacterial activity in the early stages of infection, and rapidly completing the adsorption, replication, and lysis processes. Genomic analysis reveals that R38Z possesses high genetic safety; its genome lacks virulence, resistance, and lysogen-related genes, while carrying multiple tRNA genes, which helps optimize protein translation efficiency and infection adaptability. These characteristics collectively constitute a significant advantage of R38Z in phage therapy applications.

[0017] In summary, bacteriophage R38Z possesses potential application value for Vibrio infection control in aquaculture environments. Future research can further explore its ecological stability in actual aquaculture environments, as well as its combination with other bacteriophages in cocktail formulations, leveraging its rapid lysis characteristics to provide theoretical and technical support for constructing efficient and safe bacteriophage therapy systems.

[0018] Vibrio alginolyticus phage vB_ValP_R38Z was deposited on December 8, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO: 67439-B1. Attached Figure Description

[0019] Figure 1These are morphological experimental results of bacteriophage R38Z. Figure a shows the plaques formed by R38Z; figure b shows a transmission electron microscope image of R38Z; figure c shows the adsorption rate measurement results of R38Z; and figure d shows the one-step growth curve results of R38Z.

[0020] Figure 2 This is a graph showing the results of R38Z host range determination. Figure a shows the host range of R38Z; figure b shows the results of R38Z dilution titration determination in the host.

[0021] Figure 3 This is a graph showing the environmental stability results of bacteriophage R38Z. Figure a shows the temperature stability results; figure b shows the pH stability results.

[0022] Figure 4 This is a graph showing the lysis activity results of bacteriophage R38Z.

[0023] Figure 5 This is the circular genome map of bacteriophage R38Z. The outermost circle represents the open reading frames (ORFs) encoded by the genome, with different colors indicating different functions (clockwise arrows indicate forward reading frames, and counterclockwise arrows indicate reverse reading frames); the middle blue circle represents the GC content (bulging outwards indicates a GC content higher than the genome-wide average, and concave inwards indicates the opposite); the innermost circle represents the GC offset value (G−C / G+C. Bulging outwards indicates >0, and concave inwards indicates <0).

[0024] Figure 6 This is a taxonomic diagram of bacteriophage R38Z. a) is a phylogenetic tree containing R38Z and other closely related bacteriophages, constructed using the Virus Classification and Tree Construction Online Service (VIRTOR); b) is a heatmap generated by the Virus Genome Distance Calculator (VIRDIC), combining genome similarity values ​​(right half) and alignment metrics (left half and top annotations). Detailed Implementation

[0025] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0026] Example 1:

[0027] I. Materials and Methods

[0028] 1. Isolation and purification of bacteriophages

[0029] This study used *Vibrio alginolyticus* D69_TY10 (serial number: PX700236) as the host for phage isolation. This bacterium was isolated from the culture water of *Litopenaeus vannamei* in Ningbo, Zhejiang Province (121.82981°E, 29.38545°N). This bacterium is similar to the type strain *Vibrio alginolyticus* ATCC 17749. TThe similarity was 99.79%. A 0.22 μm filter membrane (Millipore, MA, USA) was used to filter water samples to remove large particles and bacteria. The resulting filtrate was used for phage screening. The virus isolation water sample was taken from sewage at Xiamen Xiashang Aquatic Products Market (24.55°N, 118.1025°E). The host bacterium, *Vibrio alginolyticus* D69_TY10, was cultured in the dark at 28°C and 150 rpm using nutrient-rich 2216E liquid medium (10 M peptone, 2 M yeast extract, artificial seawater, pH=7.5) as the nutrient source. First, 500 μL of filtered water sample (used for phage screening) was added to the exponential growth phase Vibrio culture. The culture was incubated for 24 h in a constant temperature shaker at 28°C. The culture was then filtered through a 0.22 μm pore size filter to obtain the virus solution after removing bacteria and enriching the virus. The enriched virus solution was titrated onto the surface of a bacterial bilayer plate. The bacterial bilayer plate was prepared as follows: A lower layer plate was prepared using 2216E solid medium (1.5% agar, 10 M peptone, 2 M yeast extract, artificial seawater, pH 7.5). 5 mL of melted 2216E semi-solid medium (0.5% agar, 10 M peptone, 2 M yeast extract, artificial seawater, pH 7.5) was mixed with 1 mL of turbid host bacterial culture and poured onto the upper layer plate. The mixture was allowed to solidify to form the bacterial bilayer plate. The plates were incubated overnight at 28°C. If plaques appeared, individual plaques were picked and stored in SM buffer. The phages were then purified using the bilayer plate method, undergoing at least five rounds of purification to obtain pure phage R38Z with consistent morphological characteristics.

[0030] 2. Phage chloroform susceptibility test

[0031] To detect whether the outer capsid of bacteriophage R38Z contains lipid components or has a lipid membrane coating, samples were taken at the same concentration (~10). 8 PFU mL -1 Different volumes of chloroform (0, 20, 200, and 400 μL) were added to the R38Z phage solution, mixed well, and incubated in the dark for 30 min. The mixture was then centrifuged (5000×g, 4°C, 5 min). Finally, the morphology and number of phage plaques after chloroform treatment were analyzed using the double-layer plate method. Three parallel controls were set up for each chloroform gradient.

[0032] 3. Phage amplification and enrichment

[0033] To obtain sufficient phage for subsequent experiments, we performed phage amplification and enrichment. First, the purified phage R38Z was co-cultured with the host bacterium *Vibrio alginolyticus* D69_TY10 in a shaker at 28°C and 150 rpm in the dark, gradually scaling up to a 1 L culture system. During the culture, DNase I and RNase A were added to a final concentration of 10 mg / L to digest free nucleic acids, and NaCl was added to a final concentration of 1 mol / L to promote the release of phage from host cell debris. Subsequently, the mixture was centrifuged at 8000 × g for 15 min at 4°C, the supernatant was collected, and PEG8000 was added to a final concentration of 100 g / L. The mixture was then incubated overnight at 4°C to precipitate the phage. After centrifugation (10,000×g, 4℃, 1 h), the phage pellet was collected, washed with chloroform, and then further purified by ultracentrifugation (200,000×g, 4℃, 4 h) using a density gradient solution of CsCl (1.3, 1.5, 1.7 g / mL). Viral bands were collected. Finally, the CsCl solution was removed by centrifugation in a 30 kDa ultrafiltration tube (Merck Millipore, UFC5030BK, Germany) at 5000×g, 4℃, 5 min. The pellet was washed multiple times with SM buffer, and finally dialyzed with 30 kDa ultrafiltration tube to remove excess cesium chloride solution (5000×g, 5 min, 4℃). SM buffer was added repeatedly for washing, and finally, 2-3 volumes of SM buffer were required for dialysis.

[0034] 4. Determination of the host range of bacteriophages

[0035] Sixteen Vibrio strains were selected for host range testing. The susceptibility of isolated bacteriophages to Vibrio was determined using a dilution titration method. Vibrio strains were cultured to the logarithmic growth phase. 1 mL of the host bacterial culture was mixed with 5 mL of 0.5% 2216E semi-solid medium and poured into a lower plate (i.e., 2216E solid medium) to prepare a double-layer plate. 5 μL of a high-abundance bacteriophage R38Z suspension was dropped onto the surface of the plate, with SM Buffer used as a negative control. The growth of the phage plaques was observed at 28°C, and the host range infectable by the bacteriophages was recorded. For Vibrio strains infectable by R38Z, the infection efficiency of the bacteriophages was further determined using a dilution titration method. Finally, the host range results were visualized by constructing a 16S rDNA phylogenetic tree of the tested bacteria using the online tool Silva (Quast et al., 2013).

[0036] 5. Observation using transmission electron microscopy

[0037] Phage morphology was observed using a tungsten filament transmission electron microscope (TEM). First, the carbon support membrane was hydrophilically treated using a glow discharge apparatus to facilitate sample adsorption onto the membrane. Then, 3 μL of dialyzed and purified phage R38Z suspension was added to the carbon membrane surface and allowed to stand for 3 min before removing excess liquid. Two 5 μL aliquots of 2% (w / v) phosphotungstic acid were prepared. Once no obvious watermarks were visible on the carbon support membrane, the first aliquot was added for negative staining and immediately blotted dry. The second aliquot was then added for negative staining for 1 min, blotted dry with filter paper, and air-dried until completely dry. Finally, observation was performed using a JEM-1230 transmission electron microscope (JEOL, Tokyo, Japan) at 80.0 kV, with images acquired using a digital charge-coupled device (DC-CCP) camera.

[0038] 6. Phage gene extraction

[0039] DNA from bacteriophage R38Z was extracted using the phenol-chloroform extraction method. One mL of high-abundance bacteriophage R38Z suspension was taken, and the following solutions were added sequentially: 10 μL of proteinase K solution (20 mg / mL), 100 μL of SDS solution (10% wt / vol), and 10 μL of EDTA solution (0.5 M, pH 8.0). The mixture was thoroughly vortexed and then incubated in a 55°C metal bath for 3 h. An equal volume of a phenol-chloroform-isoamyl alcohol mixture (25:24:1 v / v) was added to the phage digest, and the mixture was centrifuged for 10 min (12,000 × g, 4°C). The supernatant was transferred to a new centrifuge tube, and this process was repeated twice. Add an equal volume of chloroform-isoamyl alcohol mixture (24:1, v / v) to the collected upper aqueous phase solution, mix well, centrifuge for 10 min (12,000×g, 4℃), transfer the upper aqueous phase solution to a new centrifuge tube, mix with an equal volume of isopropanol, incubate at -20℃ overnight to precipitate, then centrifuge for 10 min (12,000×g, 4℃). Discard the supernatant, wash the nucleic acid precipitate with 500 μL of pre-cooled 70% ethanol solution, centrifuge for 5 min (12,000×g, 4℃), repeat twice. Discard the supernatant, and after the nucleic acid precipitate dries, resuspend the DNA in 50 μL of TE buffer (10 mmol Tris-HCl, 1 mol EDTA, pH 8.0).

[0040] 7. Phage genome sequencing and analysis

[0041] The whole genome of the bacteriophage was sequenced and assembled by Shanghai Hanyu Biotechnology Co., Ltd. The probability of phage lysis was analyzed using the online analysis software phageAI (Tynecki et al., 2020). The online server GeneMarkS (Besemer et al., 2005) was used to identify putative open reading frames (ORFs) of the bacteriophage. The function of the ORFs was annotated by comparing the online BLASTP database with the non-redundant (nr) database of the National Center for Biotechnology Information (NCBI) (Altschul et al., 1997). The tRNAs in the bacteriophage genome were identified using the tRNAscan-SE v.2.0 tool (Chan et al., 2021). Furthermore, the Virulence Factor Database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD) were used to detect virulence and resistance genes in bacteriophages (Liu et al., 2022; Alcock et al., 2019) to determine whether bacteriophages contained virulence and resistance genes. A visual map of the genome's functional modules and their distribution was created using the Proksee tool. .

[0042] 8. Phage phylogenetic analysis

[0043] The complete genome sequence of bacteriophage R38Z was submitted to ViPTree (Nishimura et al., 2017), an online resource for virus classification and phylogenetic tree construction, and VICTOR (Meier-Kolthoff et al., 2022), an online service for virus classification and tree construction, to construct a phylogenetic tree including R38Z and other closely related bacteriophages, and to analyze the evolutionary relationships of the bacteriophages. Genome sequence alignment was performed using the Genome-BLAST Distance Phylogeny (GBDP) method, inferring branch support from every 100 pseudoguide replications, rooting the tree at the midpoint, and then visualizing it. A heatmap was generated using the viral genome distance calculator VIRIDIC (Moraru et al., 2020), combining genome similarity values ​​and alignment metrics.

[0044] 9. Bacteriophage Infection Kinetics Experiment

[0045] The adsorption rate of bacteriophage R38Z was determined as follows: Bacteriophages in the exponential growth phase (CFU≈1×10⁻⁶) were subjected to adsorption. 7100 mL of Vibrio alginolyticus D69_TY10 culture medium (CFU / mL) was mixed with phage R38Z suspension to control the multiplicity of infection (MOI) at 0.01. 1 mL samples were taken at 0, 5, 10, and 15 minutes, immediately centrifuged at 10,000 × g for 2 min, and the supernatant was collected for quantitative detection of unadsorbed phages. Three replicates were set up for each time point.

[0046] The one-step growth curve of bacteriophage was determined according to the method of Cai et al. (Cai et al., 2023). First, the host bacterium *Vibrio alginolyticus* D69_TY10 was cultured to the exponential growth phase, then mixed with bacteriophage R38Z suspension, controlling the multiplicity of infection to approximately 0.01–0.03. 1 mL of the mixture was taken and allowed to adsorb for 5 min at room temperature in the dark, followed by centrifugation at 8000×g for 5 min. The supernatant was discarded to remove unadsorbed bacteriophages, and this process was repeated twice. The precipitate was then resuspended in 100 mL of 2216E liquid medium, and a sample was taken at time 0 to determine the bacteriophage titer. Samples were taken every 10 min until 90 min. Three replicates were set up at each time point.

[0047] 10. Determination of phage lysis activity

[0048] Monitoring OD using a multi-functional enzyme-linked immunosorbent assay (ELISA) reader 600 (Optical density measurement at 600 nm wavelength) The growth curves of the host bacterium *Vibrio alginolyticus* D69_TY10 under different multiplicity of infection (MOI) values ​​were dynamically monitored to evaluate the lytic activity of phage R38Z against the host *Vibrio*. Freshly prepared *Vibrio alginolyticus* D69_TY10 cultures were inoculated into 96-well plates, and pre-diluted phage R38Z suspension was added. MOI gradients of 0.01, 0.1, 1, 10, and 100 were set up, along with a blank control group containing only the host bacterium. The plates were incubated at 30°C in the dark. OD was automatically measured every 30 minutes using a microplate reader. 600 Continuous monitoring was conducted for 24 hours. Eight parallel controls were set up for each MOI gradient.

[0049] 11. Environmental stability of bacteriophages

[0050] Temperature stability was assessed by measuring the change in phage titer after culturing phage suspensions of known abundance at different temperatures for the same duration. The experiment used an initial abundance of 1 × 10⁻⁶ phages. 7 Phage R38Z suspension (PFU / mL) was aliquoted into 10 mL portions and incubated at 4℃, 25℃, 37℃, 45℃, and 55℃. The number of infectious phages in the samples was detected using the double-layer plate method at preset time points of 0, 3, 24, and 48 hours. Three parallel controls were included for each treatment.

[0051] pH stability was assessed by measuring the change in phage titer after culturing phage suspensions of known abundance at different pH values ​​for the same duration. The SM buffer was first adjusted to pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 using a precision pH meter. The initial abundance was set to 1 × 10⁻⁶. 7 Phage suspensions of PFU / mL were cultured under different pH conditions. The number of infectious phages in the samples was detected using the double-layer plate method at preset time points of 0, 3, 24, and 48 hours. Three parallel controls were included for each treatment.

[0052] II. Results

[0053] 1. Morphology of bacteriophages

[0054] This study used *Vibrio alginolyticus* D69_TY10 as the host and isolated bacteriophage vB_ValP_R38Z (hereinafter referred to as R38Z) from wastewater at Xiamen Xiashang Aquatic Products Market (24.55°N, 118.1025°E) using the double-layer plate method. Culture observation showed that after 12 h of inoculation, the diameter of the bacteriophage plaques was approximately 176 ± 11 μm, with irregular, serrated edges. Figure 1 a) After 24 h of culture, the plaque diameter increased to 222 ± 12 μm; after 48 h of culture, the plaque diameter increased to 235 ± 9 μm, with serrated edges. During the 12–24 h culture period, the plaque diameter growth rate was approximately 3.8 μm / h, while during the 24–48 h culture period, the plaque diameter growth rate was approximately 1.1 μm / h. Transmission electron microscopy images showed that the phage head was icosahedral, with a head diameter of approximately 62.13 ± 2.19 nm and a tail length of approximately 19.91 ± 0.94 nm, classifying it as a typical short-tailed phage. Figure 1 b). In addition, there were no significant differences in the number and morphology of plaques in the different chloroform concentration treatment groups, indicating that the phage is not sensitive to chloroform and has no lipid membrane covering its capsid.

[0055] 2. Infection kinetics of bacteriophages

[0056] The adsorption rate of bacteriophage R38Z was measured, showing that the adsorption rate of bacteriophage R38Z reached 88% after 5 minutes of contact with the host Vibrio alginolyticus D69_TY10, and increased to 97% after 15 minutes of adsorption. Figure 1 c) indicates that phage R38Z has the ability to rapidly adsorb to the host. One-step growth curve results show that phage R38Z completes its incubation period 10 min after infecting host bacteria, enters the lysis plateau phase at 40 min, and the lysis yield is 29 PFU·cell. -1( Figure 1 d). The results showed that bacteriophage R38Z had a short incubation period and a small outbreak size.

[0057] 3. Host range of bacteriophages

[0058] The host range assay tested 16 Vibrio strains, including *V. alginolyticus*, *V. parahaemolyticus*, *V. splendidus*, *V. campbellii*, *V. neocaledonicus*, and *V. harveyi*. Plaque titration results showed that phage R38Z could only infect the isolated host *Vibrio alginolyticus* D69-TY10 (…). Figure 2 a). Further, the lytic ability of phage R38Z to infect host bacteria was determined by dilution titration. The results showed that R38Z lysed the host *Vibrio alginolyticus* D69_TY10 bacterial flora at a dilution rate of 10³ PFU / mL. -1 The abundance of these elements can form punctate plaques. Figure 2 b).

[0059] Furthermore, through experiments testing the infection of other bacteria by bacteriophage R38Z, it was found that bacteriophage R38Z could also infect another strain of Vibrio erwinii D69-TY5 isolated from the same shrimp farm water, and only when the abundance on the host Vibrio erwinii D69-TY5 reached 10... 7 PFU mL -1 Plaques may appear at times.

[0060] 4. Environmental stability of bacteriophages

[0061] The environmental stability results of phage R38Z are as follows: Figure 3 As shown. Temperature stability experiments show that ( Figure 3 a) R38Z maintains high activity within the temperature range of 4℃ to 25℃; pH stability test ( Figure 3 b) shows that its optimal survival conditions are pH 7–8, and it retains approximately 50% survival rate after 48 h of treatment within this range. Short-term treatment experiments further demonstrate that R38Z maintains some activity even under relatively alkaline conditions (pH 9–10). Overall, R38Z exhibits good stability in neutral to slightly alkaline environments and at room temperature.

[0062] 5. Lytic activity of bacteriophages

[0063] The experimental results of the determination of the lytic activity of bacteriophage R38Z against the host Vibrio alginolyticus D69_TY10 at different multiplicity of infection are shown in the figure. Figure 4 In the early stages of infection, phage R38Z exhibited strong lytic activity. All MOI groups showed OD values ​​within the first 8 hours.600 When the concentration was controlled below 0.2, bacteria in the MOI=0.01 group were rapidly lysed after a brief period of proliferation. After 9 hours of culture, the OD of the MOI=100 group... 600 The OD started to rise, while the OD of the remaining groups increased after 12 hours of incubation. 600 It began to rise gradually. Among them, MOI=1 group OD 600 The slowest rise occurred after 24 hours of incubation, with OD... 600 It remains below 0.8. Overall, R38Z exhibits excellent bactericidal ability and high lytic activity in the short term, enabling it to rapidly lyse host bacteria. This aligns with the short latency period of R38Z, meeting the need for rapid inhibition and elimination of vibrio in bacteriophage culture water.

[0064] 6. Phage genome analysis

[0065] like Figure 5 Phage R38Z has a total length of 50368 bp of linear double-stranded DNA with a GC content of 41.7%, and is predicted to contain 60 open reading frames (ORFs) and 3 tRNA genes. Of the 60 ORFs, 33 are labeled as hypothetical proteins, and 27 are annotated as proteins with known presumed functions. Excluding hypothetical proteins, the genome can be divided into four main modules: structure and assembly (11 ORFs); regulation, replication, and metabolism (6 ORFs); lysis (1 ORF); and accessory metabolic genes (8 ORFs). In the genome of phage R38Z, the three tRNA genes are located between ORF 59 and ORF 60.

[0066] Furthermore, searches of the virulence factor database and the comprehensive antibiotic resistance database revealed that phage R38Z does not contain virulence genes or antibiotic resistance genes. Phage AI software predicts that R38Z does not contain any lysogenic genes and has 100% lytic potential.

[0067] 7. Classification and genome similarity of bacteriophages

[0068] Phylogeneticly, phage R38Z and phage IME234 are on the same branch and have similar step sizes, indicating a close phylogenetic relationship. Figure 6 a). Intergenomic similarity calculated using VIRIDIC showed that R38Z had 89.7% similarity to IME234, 88.0% similarity to IME271, and less than 85% similarity to the remaining phages. Figure 6(b) According to the classification criteria of the International Committee on Taxonomy of Viruses (ICTV), a genome similarity of less than 95% is considered a new species, and less than 70% is considered a new genus. Therefore, bacteriophage R38Z should be classified as a new viral species. Source tracing analysis was performed on bacteriophages with highly similar genomes to R38Z, and a global distribution map was drawn based on the latitude and longitude information of their isolation sites. The results show that this type of bacteriophage is concentrated in the coastal areas of China and Southeast Asia.

[0069] The bacteriophage was named Vibrio alginolyticus phage (vB_ValP_R38Z) and deposited on December 8, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO: 67439-B1.

[0070] III. Discussion

[0071] Bacteriophage R38Z is characterized by rapid adsorption and a short latency period. The shorter latency period is conducive to the rapid replication and spread of bacteriophages at high bacterial densities, even with a low outbreak volume, which constitutes a competitive advantage. It can efficiently lyse the host and rapidly infect the surrounding high-density bacterial community, which provides potential application advantages for it in the control of pathogens in aquaculture.

[0072] In terms of host range, bacteriophage R38Z exhibits relatively strict host selectivity, infecting only Vibrio isolated from shrimp farming water. This specificity is beneficial for targeted inhibition of pathogenic Vibrio in aquaculture environments, reducing interference with beneficial microbial communities, lowering the risk of non-targeted transmission, and thus improving its safety and eco-friendliness in aquaculture environments.

[0073] At the molecular level, R38Z encodes two tail fibrous proteins (ORF46 and ORF48) and one endolysin (ORF57). ORF46 contains a disordered region at its C-terminus, enhancing its adaptability to binding to the host surface. ORF48 possesses an ICA domain, responsible for the proper folding, trimerization, and autocatalytic maturation of the tail fibrous material, ensuring recognition stability. ORF57 is a glycoside-hydrolyzing endolysin that specifically hydrolyzes peptidoglycan, synergistically promoting rapid cell lysis with outer membrane permeability proteins. These characteristics collectively constitute the basis for R38Z's highly efficient infection and lysis processes.

[0074] Furthermore, R38Z carries multiple accessory metabolic genes (AMGs), including tRNA nucleotidyltransferases, glutamine aminotransferases, and several tRNA genes (Ile, Pro, Ser). These genes help maintain tRNA function during infection, optimize nitrogen metabolic flux, and compensate for codon usage bias, thereby supporting efficient phage protein synthesis and replication. This genetic configuration enhances R38Z's metabolic adaptability and translational autonomy within the host, providing a molecular basis for its potential therapeutic potential.

Claims

1. Vibrio alginolyticus phage ( Vibrio alginolyticus phage)vB_ValP_R38Z, accession number: GDMCC NO: 67439-B1.

2. A microbial preparation for controlling Vibrio alginolyticus or Vibrio erwinii, characterized in that, It contains the bacteriophage vB_ValP_R38Z as described in claim 1 as an active ingredient.

3. The use of the bacteriophage vB_ValP_R38Z of claim 1 or the microbial preparation of claim 2 in the prevention and / or killing of Vibrio alginolyticus or Vibrio erwinis.

4. The use of the bacteriophage vB_ValP_R38Z of claim 1 or the microbial preparation of claim 2 in the preparation of products for the prevention and / or killing of Vibrio alginolyticus or Vibrio erwinis.

5. The application according to claim 4, characterized in that, The Vibrio alginolyticus and Vibrio erwinii mentioned above are from the water used for Litopenaeus vannamei farming.

6. The application according to claim 4, characterized in that, The alginolytic Vibrio is Vibrio alginolyticus D69-TY10.

7. The application according to claim 4, characterized in that, The products mentioned include pharmaceuticals, disinfectants, water purifiers, feed or feed additives.

8. A product for controlling Vibrio alginolyticus or Vibrio erwinii infection, characterized in that, The preparation contains the bacteriophage vB_ValP_R38Z of claim 1 or the microbial preparation of claim 2.

9. The product according to claim 9, characterized in that, The alginolytic Vibrio is Vibrio alginolyticus D69-TY10.

10. A method for in vitro prevention and control of harmful infections caused by Vibrio alginolyticus or Vibrio erwinii, characterized in that, It utilizes the lysis effect of bacteriophage vB_ValP_R38Z as described in claim 1 or the microbial preparation as described in claim 2 on Vibrio alginolyticus or Vibrio erwinis to kill Vibrio alginolyticus or Vibrio erwinis in vitro.

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