Vibrio campestris phage vB_VcaS_R45Z and its applications

Abstract

The application discloses a Vibrio camberensis phage vB_VcaS_R45Z and application thereof. Vibrio campbellii The phage vB_VcaS_R45Z is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on December 8, 2025, is located at No. 100, Martyr's Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and has a postal code of 510070, and has a preservation number of GDMCC NO.67441-B1. The phage vB_VcaS_R45Z has rapid lysis ability, high host inhibition effect, safe genome structure and clear systematic classification position, and provides an important basis for the phage as a biological control factor of aquatic vibrio pathogen.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to Vibrio campbellii bacteriophage vB_VcaS_R45Z and its applications. Background Technology

[0002] In recent years, with the continuous advancement of aquaculture technology, China's aquatic product production model has been rapidly shifting from the traditional "fishing-based" to "aquaculture-based." New types of facility-based fisheries, such as deep-sea aquaculture, land-based recirculating aquatic systems (RAS), and aquaponics, have developed rapidly, providing crucial support for the large-scale and green development of aquaculture and driving continuous growth in aquaculture output. However, high-density, intensive aquaculture models have exacerbated eutrophication and microecological imbalances in water bodies, providing favorable conditions for the proliferation of pathogenic microorganisms. Rapid pathogen proliferation is a significant cause of disease outbreaks in aquaculture.

[0003] In aquaculture systems, Vibrio is one of the most common and damaging pathogenic microorganisms. This genus of bacteria is widely distributed in marine, estuarine, and brackish water environments, and thrives particularly well in warm, eutrophic waters. To date, over 100 species of Vibrio have been identified, many of which are closely associated with aquatic animal diseases, including *Vibrio parahaemolyticus*, *Vibrio alginolyticus*, *Vibrio vulnificus*, *Vibrio campbellii*, *Vibrio harveyi*, and *Vibrio owensii*. These pathogenic Vibrios can spread rapidly through water, feed, biofilms, or co-cultured animals, causing various diseases such as septicemia, ulceration, enteritis, bioluminescence, and acute hepatopancreatic necrosis, posing a serious threat to the health of shrimp, crabs, shellfish, and fish. Summary of the Invention

[0004] The purpose of this invention is to provide Vibrio campestris phage vB_VcaS_R45Z and its applications.

[0005] The first objective of this invention is to provide Vibrio campbellii phage vB_VcaS_R45Z, which 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. 67441-B1.

[0006] A second objective of this invention is to provide a microbial preparation for controlling Vibrio campbellii or Vibrio anguillarum, which contains the aforementioned bacteriophage Vibrio campbellii phage vB_VcaS_R45Z as an active ingredient.

[0007] A third objective of this invention is to provide the use of the aforementioned bacteriophage Vibrio campbellii phage vB_VcaS_R45Z or microbial preparations in the prevention and / or eradication of Vibrio campbellii or Vibrio anguillarum.

[0008] A fourth object of the present invention is to provide the use of the above-mentioned bacteriophage Vibrio campbellii phage vB_VcaS_R45Z or microbial preparation in the preparation of products for the prevention and / or killing of Vibrio campbellii or Vibrio anguillarum.

[0009] Preferably, the Vibrio campbellii is Vibrio campbellii JL2918.

[0010] Preferably, the Vibrio anguillarum is Vibrio anguillarum LMG 4437. T

[0011] Preferably, the products include pharmaceuticals, disinfectants, and water purifiers.

[0012] A fifth object of the present invention is to provide a product for controlling Vibrio campbellii or Vibrio anguillarum infection, which contains the aforementioned bacteriophage Vibrio campbellii phage vB_VcaS_R45Z or a microbial preparation.

[0013] The sixth objective of this invention is to provide a method for preventing harmful infections of Vibrio campbellii or Vibrio anguillarum in vitro. This method utilizes the lysis of Vibrio campbellii or Vibrio anguillarum by the aforementioned bacteriophage Vibrio campbellii phage vB_VcaS_R45Z or microbial preparations to kill Vibrio campbellii or Vibrio anguillarum in vitro.

[0014] This invention systematically elucidates the biological characteristics, lysis kinetics, genomic structure, and taxonomic position of bacteriophage R45Z against Vibrio campbellii JL2918. Lysis kinetics results show that R45Z can induce lysis in the early stages of bacterial proliferation at different infectious doses, exhibiting significant MOI-dependent and time-dependent antibacterial effects. The high MOI group showed a significant decrease in bacterial density 3–4 h post-infection, while the medium- and low MOI groups, although exhibiting weaker antibacterial activity, maintained effective inhibition of the host in the later stages of infection. One-step growth curves show that R45Z has a latency period of approximately 20 min and a high outbreak rate, indicating its rapid replication and high lysis efficiency. Whole-genome sequencing results show that R45Z is a double-stranded DNA bacteriophage with a genome length of 59,278 bp, predicting 87 ORFs, of which 38 have definite or inferred functions, and the remainder are hypothetical proteins. The genome contains a complete structural assembly gene module and a lysis module composed of membrane proteins and N-acetylmuramoyl-L-alanine amidase, and no resistance genes or virulence factors were detected, suggesting that it is a safe lytic phage. Furthermore, the genome contains a proline-specific tRNA gene, which may be related to optimized viral protein translation. Phylogenetic and genome similarity analysis showed that R45Z clustered with vB_VpS_CA8 and vB_VpaS_AL2 in the same branch, but their genome similarity was less than 95%, meeting the ICTV criteria for species classification. Therefore, it can be classified as a new species phage within the Queuovirinae subfamily. In summary, R45Z's rapid lysis ability, high host inhibition effect, safe genome structure, and clear taxonomic position provide an important basis for its role as a biocontrol agent for Vibrio aquatic pathogens.

[0015] Vibrio campbellii phage vB_VcaS_ R45Z 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. 67441-B1. Attached Figure Description

[0016] Figure 1 This study examines the morphological characteristics of bacteriophage R45Z and its infection dynamics in the host. (a) Morphology of plaques of bacteriophage R45Z after incubation with V. campbellii JL2918 for 7 h and 28 h; (b) Transmission electron microscopy morphology of bacteriophage R45Z.

[0017] Figure 2This is the host kinetics of bacteriophage R45Z. (a) One-step growth curve of R45Z (n = 3); (b) Lysis curve of bacteriophage R45Z (n = 8).

[0018] Figure 3 This is a circular map of the entire genome of bacteriophage R45. The map shows the complete genome structure and functional annotation of bacteriophage R45. The outermost circle shows the genomic backbone (contigs); the second and third circles represent gene locations on the positive and negative strands of the R45 genome, respectively, with different colored arrows indicating different functional categories.

[0019] Figure 4 This involves phylogenetic analysis and whole-genome alignment of bacteriophages. (a) Phylogenetic tree, generated based on the Virus Classification and Tree Construction Online Resource (VICTOR), constructed with reference to the NCBI RefSeq database. Pairwise comparisons of amino acid sequences were performed using the Genomic Distance Phylogenetic (GBDP) method. The numbers in the top branch are GBDP pseudo-guided support values ​​from 100 replications; (b) Heatmap generated by the inter-genome distance calculator (inter-virus distance), including inter-genome similarity values ​​(right half) and alignment metrics (left half and top annotations). Detailed Implementation

[0020] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0021] Example 1:

[0022] 1. Materials and Methods

[0023] 1.1 Isolation and purification of bacteriophages

[0024] The host strain used in this study was *V. campbellii* JL2918 (NCBI serial number PX745080, which the applicant also holds and guarantees to make available to the public for 20 years from the date of application). Water samples were collected in December 2022 from wastewater pipes at the Shekou Seafood Market in Nanshan District (113°55′12″ E, 22°31′33″ N) for phage isolation. The water samples were filtered through a 0.22 μm polycarbonate membrane (Polycarbonate, Millipore, USA) and stored in a laboratory refrigerator at 4°C.

[0025] This experiment employed the double-layer plate method for the isolation and purification of bacteriophages. Three mL of *V. campbellii* JL2918 in the logarithmic growth phase was co-cultured with 100 μL of wastewater filtrate for one day to enrich any potential bacteriophages present. Subsequently, the collected culture was sterilized by filtration through a 0.22 μm polycarbonate membrane and serially diluted with autoclaved SM buffer before being mixed with logarithmic growth phase host bacteria to prepare double-layer plates. The plates were incubated at 37°C, and plaque formation was observed periodically. Once clear, well-defined plaques appeared, they were transferred to SM buffer and stored at 4°C. To obtain pure bacteriophages, at least five double-layer plate isolation and purification cycles were performed to obtain a bacteriophage suspension. The size and morphological changes of the plaques were observed and recorded using a plaque imaging device (SONY ILCE-7RM3A).

[0026] 1.2 Preparation of high-abundance phage suspensions

[0027] The purified phage suspension was filtered through a 0.22 μm filter membrane to remove residual cells and impurities. 500 μL of the phage filtrate was inoculated into 20 mL of logarithmic growth phase V. campbellii JL2918 culture medium. The above steps were repeated to recover the filtrate and expand the phage culture to a 1 L system. A final concentration of 1 μg / mL was added to 1 L of host-phage expansion medium. -1 DNase I and RNase A were digested in the culture medium at room temperature in the dark for 1 h to degrade free DNA and RNA. Then, 1 mol / L NaCl was added, and after complete dissolution, the medium was incubated at 4°C for 1 h to promote phage detachment from fragments. The supernatant was collected by centrifugation (10,000 × g, 4°C, 15 min) and filtered through a 0.22 μm filter to obtain 1 L of phage filtrate. PEG 8000 (final concentration 100 g / L) was added to 1 L of the phage filtrate, and the medium was incubated at 4°C in the dark for 1–3 days. After significant flocculent sedimentation, the medium was centrifuged (12,000 × g, 4°C, 1 h), the supernatant was discarded, and the precipitate was resuspended in 6 mL of SM buffer. Following the guidelines in *Molecular Cloning: A Laboratory Manual*, the resuspended phage was purified using CsCl density gradient centrifugation. After stratifying into density bands of 1.3, 1.5, and 1.7 g / mL, the phage resuspension was added, and the mixture was centrifuged at 200,000 × g for 6 h at 4°C using an SW 32 Ti rotor (Optima X-100 ultracentrifuge, Beckman Coulter, USA). Phage particles were collected from the 1.5 g / mL density band and dialyzed to remove salts using a 30 kDa ultrafiltration tube (Millipore, Bedford, MA, USA) for subsequent experiments. This yielded phage R45Z.

[0028] 1.3 Morphological observation by transmission electron microscopy

[0029] This study used negative staining to observe phage morphology. 5 μL of phage with an infectivity of 10-1 was collected. 10 PFU mL -1 The concentrated phage R45Z solution was dropped onto a 200-mesh gold-sprayed copper grid and allowed to adsorb in the dark for 3-5 min. It was then negatively stained twice with 2% phosphotungstic acid, and after each staining, it was allowed to air dry for 3 min. In this study, images of the samples were observed using a tungsten filament transmission electron microscope (HITACH HT7700, Hitachi, Ltd., Japan) at 120 kV.

[0030] 1.4 Determination of phage adsorption efficiency

[0031] Phage adsorption rate is an indicator of the efficiency of phage binding to receptors on the surface of host bacteria, and it directly affects the phage infection process. At the optimal MOI ratio (0.01), the diluted phage R45Z suspension was mixed with the exponentially growing host bacterium *V. campbellii* JL2918. Immediately afterwards, 1 mL was transferred to a 1.5 mL centrifuge tube, centrifuged at 12000 × g for 1 min, and 100 μL of the supernatant (containing free phage) was serially diluted and the abundance was detected on bilayer plates. The above steps were repeated every 5 min. The experiment was conducted in triplicate, and the average value was taken as the final result.

[0032] 1.5 One-step growth curve of bacteriophage

[0033] One-step growth curves of bacteriophages are a standard method for characterizing the growth cycle of lytic bacteriophages, with latency and lysis rate typically used as the main evaluation indicators. At the optimal MOI ratio (0.01), diluted bacteriophage R45Z suspension was mixed with host bacterium *V. campbellii* JL2918, allowed to stand at room temperature for 5 min for adsorption, and then centrifuged (8000 × g, 4℃, 5 min) to remove unadsorbed bacteriophages. The precipitate was resuspended in 1 mL of 2216E medium and then added to 100 mL of medium (denoted as T0 = 0), and incubated at 37℃. Samples (100 μL) were taken every 10 min, and titers were determined using the double-layer plate method. The experiment was conducted in triplicate, and the average value was taken as the final result.

[0034] 1.6 Determination of bacteriophage bactericidal efficiency

[0035] To evaluate the lytic activity of bacteriophages against V. campbellii JL2918, this study designed bacteriophage bactericidal experiments under different MOI conditions. The initial logarithmic growth phase (OD) was used to determine the lytic activity of bacteriophages against V. campbellii JL2918. 600nmHost bacteria (0.10–0.20 μO) were inoculated at a 1:50 ratio into 2216E liquid medium and cultured with shaking at 30°C and 160 rpm / min. After the bacteria reached the predetermined OD value, phage R45Z was inoculated at MOIs of 0.01, 0.1, 1, 10, and 100 to construct a Vibrio-phage co-incubation system. The mixture was then aliquoted into 96-well plates (200 μL per well) and incubated at 28°C for 24 h using a multi-functional microplate reader (SynergyH1, BioTek, USA). The absorbance was monitored in real-time at 600 nm. 600 ), and was tested every 30 minutes. Eight biological replicates were set up for each treatment, with 2216E medium as a negative control and bacterial suspension without phage as a positive control.

[0036] 1.7 DNA Extraction and Sequencing

[0037] DNA from phage R45Z was extracted using the phenol-chloroform-isoamyl alcohol method. 1 mL of purified phage solution was mixed with proteinase K (10 μL), SDS (100 μL), and EDTA (10 μL), and incubated at 55 °C for 3 hours to lyse the phage capsid. An equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) was then added, mixed, and centrifuged (12,000 × g, 4 °C, 10 min). The supernatant was collected, and this process was repeated twice. An equal volume of chloroform-isoamyl alcohol (24:1) was added, mixed, and centrifuged to remove residual phenol. An equal volume of isopropanol was added to the aqueous phase, and the mixture was incubated overnight at -20 °C. The DNA was then precipitated by centrifugation, washed twice with 70% ethanol, dried, and dissolved in 50 μL TE buffer. The concentration and purity of the DNA (A260 / A280 ratio 1.8–2.0) were determined by electrophoresis and NanoDrop assay, and the DNA was stored at -20 °C.

[0038] Phage whole-genome sequencing was performed by Shanghai Hanyu Biotechnology Co., Ltd. (China). The whole-genome sequencing was conducted using the Illumina Nova platform. 1 μg of DNA was fragmented to approximately 300 bp using a Covaris M220 sonicator. A library was constructed using the NEBNext® Ultra™ DNA Library Prep Kit, including end repair, adapter ligation, and PCR amplification. The final purified library contained high-quality inserts of 300-500 bp. The library was then sequenced end-to-end using the TruSeq PE Cluster Kit on an Illumina Nova instrument, generating 2 × 150 bp data.

[0039] 1.8 Genomics Analysis

[0040] Raw sequencing data were processed using Trimmomatic v0.32 to remove adapter sequences, filter low-quality bases and short sequences, resulting in Clean Reads. De novo assembly was performed using Velvet v1.2.03, and the generated genome sequences were saved in FASTA format. After the phage R45Z genome was assembled, its genetic function and ecological safety were annotated and evaluated using various bioinformatics tools. Open Reading Frames (ORFs) prediction was performed using GeneMarkS software (Besemer et al., 2001). Subsequently, the BLASTP tool (threshold: E-value ≤ e) was used. -5 The predicted ORFs were compared with the NCBI Non-Redundant Protein Database (NR Database) to annotate functional and structural proteins (Huerta-Cepas et al., 2017).

[0041] Subsequently, tRNA genes in the genome were detected using tRNAscan-SE software (Schattner et al., 2005), and viral sequences were identified and classified using VirSorter2. The results were then converted into a format suitable for DRAM analysis. Genome regions were annotated using DRAM software, with a focus on identifying the functions and metabolic pathways of auxiliary metabolic genes (AMGs).

[0042] In terms of ecological safety assessment, the Virulence Factors Database (VFDB) and the Antibiotic Resistance Gene Database (ARGD) were used to screen for potential toxicity genes and antibiotic resistance genes in the genome (Alcock et al., 2020; Chen et al., 2005). Visualization maps of genomic functional modules and their distribution were generated using the Proksee tool (Grant et al., 2023).

[0043] 1.9 Phage phylogenetic and taxonomic analysis

[0044] Intergenomic similarity analysis was performed using Viral CONTigs Automatic Clustering and Taxonomy v2.0 (vConTACT2), comparing phages with high similarity based on gene-sharing characteristics with the Prokaryotic Viral RefSeq 207 database. The complete genome sequences of the phages were also submitted to NCBI BLASTN for further searching for similar sequences.

[0045] The evolutionary relationships of bacteriophages were constructed using the online tool Virus Classification and Tree Building (VICTOR). Genome-BLAST Distance Phylogeny (GBDP) was used for pairwise amino acid sequence comparisons, and a phylogenetic tree was generated under recommended settings. The tree was then visualized using the ggtree package (Meier-Kolthoff & Göker, 2017; Yu et al., 2017). Furthermore, the genomic nucleotide similarity and the proportion of aligned genomic fragments among bacteriophages were calculated using Virus Intergenomic Distance Calculator (VIRIDIC) (Moreno-Gallego et al., 2019).

[0046] 2 Results

[0047] 2.1 Phage morphological characteristics

[0048] This study used *V. owensii* JL2918 as the host and obtained a bacteriophage strain, named R45Z, from wastewater in a seafood market. Bacteriophage R45Z formed circular, indistinct plaques with well-defined edges on double-layer plates, without surrounding halos, consistent with the typical characteristics of lytic bacteriophages. To understand the formation and diffusion dynamics of these plaques in detail, this study observed them at different culture time points. Figure 1 a). After 7 hours of incubation, plaques initially formed, with a diameter of approximately 0.27 ± 0.09 mm. After 28 hours of incubation, the plaque diameter did not change significantly, but the plaques became more transparent, with clear edges and no surrounding halo. Purified phage R45Z was observed by TEM; R45Z exhibited typical long-tailed phage structural features, consisting of an icosahedral head: head length 91 ± 2.92 nm, head width 63 ± 1.40 nm, and a tail length of approximately 135 ± 16.55 nm. Figure 1 b).

[0049] 2.2 Phage Infection Dynamics

[0050] Analysis of the one-step growth curve of bacteriophage R45Z showed that its infection cycle can be divided into three stages: latent period, outbreak period, and plateau period. Figure 2a) The latency period of bacteriophage R45Z is approximately 20 minutes, during which the phage undergoes genome replication, protein synthesis, and progeny phage assembly. Subsequently, the phage rapidly lyses, entering a burst phase until reaching a plateau phase at 40 minutes. Experimental results show that the lysis rate of R45Z is approximately 374 ± 30 PFU / cell, indicating that a single cell can produce a high number of progeny phages during a single replication cycle. These results demonstrate that bacteriophage R45Z has a short latency period and a high lysis rate, highlighting its efficient replication and lysis capabilities.

[0051] This study compared the lysis kinetics of phage R45Z on V. campbellii JL2918 under different MOI conditions. Figure 2 b). In the control group (gray squares) uninfected with bacteriophages, bacteria showed a continuous growth trend, with OD values ​​increasing after 8 hours. 600 The concentration rose to approximately 1.4 and remained stable throughout the 24-hour incubation period. Meanwhile, all phage-infected groups showed OD values ​​in the early stages of culture. 600 The decrease in OD values ​​indicates that bacteriophages can initiate lysis during the initial bacterial proliferation stage. The antibacterial effect of bacteriophages increases with increasing MOI. High MOI (10 and 100) groups showed the most significant inhibitory effect, with OD values ​​remaining elevated 3–4 h post-infection. 600 The MOI decreased to approximately 0.15–0.20, then began to slowly recover after 8–10 h. Moderate MOIs (1 and 0.1) also significantly reduced bacterial density, but the lowest point was approximately 0.25–0.35, showing slightly weaker inhibitory effects than the high MOI group. The lowest MOI (0.01) only produced partial lysis, and bacterial growth recovered earlier than in other infection groups. All infection groups showed varying degrees of regrowth after approximately 10 h, but the recovery rate and final OD differed. 600 The values ​​were all negatively correlated with the MOI of infection. In summary, R45Z exhibited outstanding MOI and time-dependent bactericidal properties at different infectious doses and durations of action.

[0052] 2.3 Determination of the host range of bacteriophages

[0053] The 23 Vibrio strains tested in this study included *Vibrio owensii*, *Vibrio parahemolyticus*, *Vibrio harveyi*, *Vibrio alginolyticus*, *Vibrio campbellii*, *Vibrio splendidus*, *Vibrio anguillarum*, *Vibrio diabolicus*, and *Vibrio fluvialis*. The host range results are shown in Table 1. Bacteriophage R45Z was able to lyse *Vibrio campbellii* JL2918, two *Vibrio owensii* strains XZY4 and XZY43, and one *Vibrio anguillarum* strain LMG 4437. T .

[0054] Table 1. Host range of bacteriophage R45Z

[0055] V. owensii XZY2 - V. owensii XZY4 + V. owensii XZY5 - V. owensii XZY6 - V. owensii XZY42 - V. owensii XZY43 + V. owensii XZY45 - V. owensii XZY46 - V. parahemolyticus <![CDATA[ATCC 17802 T ]]> - V. parahemolyticus Vp-201911 - V. parahemolyticus Pir-201806 - V. parahemolyticus HL-202005 - V. harveyi <![CDATA[LMG 4044 T ]]> - V. alginolyticus <![CDATA[ATCC 17749 T ]]> - V. alginolyticus BVA2 - V. alginolyticus HJ-1 - V. alginolyticus HJ-2 - V. campbellii LMG 11216 - V. campbellii JL2918 + V. campbellii VSP1 - V. splendidus <![CDATA[ATCC 33125 T ]]> - V. anguillarum <![CDATA[LMG 4437 T ]]> + V. diabolicus WPAGA3 - V. fluvialis <![CDATA[ATCC 33810 T ]]> -

[0056] 2.4 Genomic characteristics of bacteriophage R45Z

[0057] like Figure 3 As shown, the genetic material of bacteriophage R45Z is double-stranded DNA (dsDNA), with a genome length of 59,278 bp and a G+C content of 46.37%. A total of 87 open reading frames (ORFs) were predicted in the R45Z genome, of which 38 ORFs have definite or inferred functions, and the remaining 49 (56.3%) were annotated as hypothetical proteins. The annotated genes mainly involve DNA replication and nucleotide metabolism, viral particle structure and assembly, DNA packaging, host cell lysis, and auxiliary metabolic functions. Tail assembly proteins, head structural proteins, and terminase genes are continuously distributed, consistent with the typical genomic organization characteristics of long-tailed dsDNA bacteriophages. Furthermore, a complete lysis module composed of membrane proteins and N-acetylmuramoyl-L-alanine amidase was identified in the genome, indicating that this bacteriophage has a lysis-type life cycle. Further analysis revealed one tRNA gene predicted in the phage R45 genome. This tRNA is a proline-specific tRNA (tRNA-Pro), with the anticodon TGG, located in the 13,061–13,137 bp region of the genome, and is 77 bp in length. No introns were detected, and the prediction confidence was high (score = 84.3). The presence of this tRNA may help alleviate the restriction of specific codons in the host translation system during infection, thereby improving the translation efficiency of viral proteins. Furthermore, no genes associated with antibiotic resistance or virulence factors were detected in the genome.

[0058] 2.5 Classification and genomic similarity of bacteriophage R45Z

[0059] To investigate the phylogenetic position of bacteriophage R45Z, this study first conducted genome-wide homology analysis. The R45Z genome was aligned using NCBI BLASTN (v2.14.0+), with selection criteria set as: Percentage Identity (Per. Ident) > 85% and a significance threshold E = 0.00001. Finally, 39 highly homologous bacteriophage sequences were obtained from the RefSeq database. Phylogenetic analysis results showed ( Figure 4 a) R45Z is in the same subfamily as vB_VpS_CA8 (CA8) and vB_VpaS_AL2 (AL2). Based on current phage classification standards and various analysis results, R45Z is confirmed to belong to the subfamily Queuovirinae. To better estimate the similarity between phage genomes, all phages in the phylogenetic tree were imported into the VIRIDIC software and classified according to similarity thresholds of <95% for new species and <70% for new genera. The results showed that R45Z had sequence similarities of 89.1% with CA8 and 89.6% with AL2, both less than 95%, therefore R45Z is a new species ( Figure 4 b).

[0060] The bacteriophage R45Z was named *Vibrio campbellii phage vB_VcaS_R45Z*, and 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. 67441-B1.

[0061] 3. Discussion

[0062] This study systematically evaluated the biological characteristics and lysis kinetics of the lytic bacteriophage R45Z against Vibrio campbellii JL2918. One-step growth curves showed that R45Z had a short latency of approximately 20 min and a large outbreak rate of 374 ± 30 PFU / cell, indicating that this bacteriophage possesses the ability to rapidly replicate and efficiently release progeny particles, suggesting that R45Z has the potential to serve as a rapid antibacterial agent in aquatic environments.

[0063] MOI lysis kinetics further revealed that the bactericidal effect of R45Z on V. campbellii JL2918 was significantly dose- and time-dependent. The control group continued to grow throughout the 24 hours, while all phage-infected groups showed OD (disruptive growth) in the early stages of infection (approximately 2–4 hours). 600 The significant decrease indicates that the phage can initiate lysis during the initial bacterial proliferation stage. Furthermore, the presence of both membrane proteins and N-acetylmuramoyl-L-alanine amidase in the lysis module encoded by the R45Z gene enhances its characteristics as a lytic phage, facilitating its rapid antibacterial action in real-world environments. However, it is noteworthy that varying degrees of bacterial regrowth occurred in all infection groups after 10 hours. This phenomenon may stem from the following mechanisms: (i) a transient decrease in phage concentration in the culture system after the initial large-scale lysis, allowing a small number of uninfected or surviving hosts to proliferate again; and (ii) transient tolerance or phenotypic variation in host cells under selective pressure. Similar regrowth phenomena have been reported in various Vibrio-phage systems, and bacterial resurgence can usually be further suppressed by using phage cocktails or in combination with antimicrobial compounds. Despite regrowth, the overall bacterial density remained low at 24 hours, indicating that R45Z possesses sustained and significant lytic capabilities.

[0064] In summary, R45Z possesses a clear phylogenetic position and a strictly cleaved genome. No antibiotic resistance genes, host virulence factors, integrases, or lysogen-related genes were detected in its genome, indicating high biosafety and meeting the basic requirements for its use as a biocontrol tool for aquatic diseases. This lays a solid foundation for its application in pathogen control in aquaculture.

Claims

1. Vibrio campei bacteriophage Vibrio campbellii Phage vB_VcaS_ R45Z, accession number: GDMCCNO. 67441-B1.

2. A method for controlling Vibrio anguillarum ( Vibrio anguillarum The microbial preparation of ) is characterized in that, Contains the bacteriophage according to claim 1 Vibrio campbellii Phage vB_VcaS_R45Z is used as the active ingredient.

3. The bacteriophage according to claim 1 Vibrio campbellii Phage vB_VcaS_R45Z or the microbial preparation of claim 2 for the prevention and / or eradication of Vibrio anguillarum LMG 4437 for non-disease diagnostic and therapeutic purposes. T Applications in [the field].

4. The bacteriophage according to claim 1 Vibriocampbellii Phage vB_VcaS_R45Z or the microbial preparation according to claim 2 in the preparation of prevention and / or killing of Vibrio anguillarum LMG 4437 T Applications in the product.

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

6. A product for controlling Vibrio campestris infection, characterized in that, Contains the bacteriophage according to claim 1 Vibrio campbellii Phage vB_VcaS_R45Z or the microbial preparation according to claim 2.

7. A method for the in vitro prevention and treatment of harmful Vibrio anguillarum infections for non-disease diagnostic and therapeutic purposes, characterized in that, It utilizes the bacteriophage described in claim 1 Vibrio campbellii Phage vB_VcaS_R45Z or the microbial preparation of claim 2 against Vibrio anguillarum LMG 4437 T The lytic activity of [the substance] kills Vibrio anguillarum LMG 4437 in vitro. T .

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