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. 2026 Oct 7;373:199813. doi: 10.1016/j.virusres.2026.199813

Characterization and genomic analysis of a jumbo Aeromonas hydrophila phage BMBAH1, representing a candidate novel viral genus

Shitian Huo a,b,c,d, Pengfei Zhu a,b,c,d, Jikai Xu a,b,c,d, Kai Wang a,b,c,d, Ming Sun a,b,c,d, Donghai Peng a,b,c,d,⁎
PMCID: PMC13678102  PMID: 42843456

Highlights

  • •

    A novel jumbo phage, BMBAH1, infecting Aeromonas hydrophila was isolated and characterized.

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    Phage BMBAH1 exhibited broad temperature and pH stability, a large burst size, and sustained antibacterial activity.

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    Phage BMBAH1 possesses a 237 kb genome encoding 241 predicted ORFs, many of which remain functionally uncharacterized.

  • •

    Comparative genomic and phylogenetic analyses assigned phage BMBAH1 to the family Chimalliviridae.

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    Phage BMBAH1 represents the founding member of a candidate novel viral genus, Southlakevirus.

Keywords: Aeromonas hydrophila, Jumbo phage, Genomic characterization, Phylogenetic analysis, Southlakevirus

Abstract

Aeromonas hydrophila is an opportunistic pathogen of fish, humans, and livestock, and has a severe negative impact on aquaculture development. Phage therapy is considered an alternative strategy for controlling bacterial infections and contamination. In this study, a novel phage, Aeromonas hydrophila BMBAH1, was isolated. Transmission electron microscopy showed that phage BMBAH1 had a long tail and an icosahedral head. Biological characterization indicated that phage BMBAH1 remained highly active across a broad range of temperatures and pH values, exhibiting long-term and significant inhibitory effects on host bacteria at an optimal multiplicity of infection (MOI) of 0.1. The genome of phage BMBAH1 is 237 kb in length with a guanine-cytosine (GC) content of 44.3%, and it was identified as a typical jumbo phage and predicted to contain 241 open reading frames (ORFs). Whole-proteome-based phylogenetic analysis, gene-sharing network analysis, and core protein clustering consistently indicated that phage BMBAH1 belongs to the family Chimalliviridae. However, phage BMBAH1 exhibited low intergenomic similarity to previously isolated phages, with values of approximately 30%. These results support the designation of phage BMBAH1 as the representative member of a candidate novel viral genus, designated Southlakevirus. Our findings expand the diversity of A. hydrophila phages and provide a promising genetic resource for further exploring biological characteristics of jumbo phages and developing biocontrol strategies.

1. Introduction

Aeromonas hydrophila belongs to the genus Aeromonas of the family Aeromonadaceae. It is a Gram-negative opportunistic pathogen widely distributed in freshwater ecosystems, aquatic products, and food-processing environments (Barbara and Francesca, 2015). This bacterium exhibits strong environmental adaptability, and it can survive across a broad range of temperatures, pH values, and salinity conditions. Moreover, its tolerance to adverse environments and antibiotics can be enhanced through biofilm formation (Awan et al., 2018; Del Castillo et al., 2013; Nagar et al., 2016). A. hydrophila also produces a variety of virulence factors, including hemolysins, extracellular enzymes, and secretion systems, playing important roles in host infection and pathogenesis (Harshitha et al., 2023).

As one of the major pathogens in freshwater aquaculture systems, A. hydrophila can infect a wide range of aquatic animals, including fish, crustaceans, and reptiles, causing substantial economic losses worldwide (Barger et al., 2021). In addition, this bacterium can be transmitted through water or aquatic food products, infecting humans and causing diseases such as gastroenteritis and septicemia, thereby posing a public health risk (Awan et al., 2018; Thayumanavan et al., 2003). Currently, the prevention and control of A. hydrophila infections mainly rely on antibiotics. However, the long-term and excessive use of antibiotics has resulted in the rapid emergence of antibiotic-resistant strains, severely limiting antibiotic effectiveness (Daskalov, 2006; Dong et al., 2021; Nithin et al., 2021). Therefore, the development of safe, effective, and environmentally friendly alternative control strategies has become an urgent need.

Phages are viruses that can specifically infect and lyse bacteria and have attracted attention as alternatives or complements to antibiotics. Compared with antibiotics, phages exhibit multiple advantages, including strong host specificity, effectiveness against antibiotic-resistant strains and biofilms, and minimal impact on the environment (Jun et al., 2013; Kokkari et al., 2018). In recent years, phages have been extensively studied and applied in multiple fields such as human disease, food safety, and aquaculture (Hall et al., 2012). Some studies have been conducted to isolate A. hydrophila phages and investigate their bactericidal activity (Paterson et al., 1969; Wang et al., 2024). Interest in phage therapy has continued to increase, and some phage preparations have been approved by the U.S. Food and Drug Administration (FDA) and successfully commercialized (Pinto et al., 2020; Ramos-Vivas et al., 2021). Based on genome size, phages with genomes larger than 200 kb are classified as jumbo phages (Yuan and Gao, 2017). Some jumbo phages carry additional functional genes, exhibiting the potential to counteract bacterial defense systems, and thus they are of great significance in phage evolution and applied research (Nazir et al., 2021). For example, some jumbo phages specifically encode the tubulin-like protein PhuZ, which contributes to the formation of a nucleus-like compartment. This structure spatially organizes phage DNA within the host cell and may help limit the access of certain host defense systems to phage genetic material (Guan and Bondy-Denomy, 2020). Compared with conventional phages, some jumbo phages may possess enhanced genetic and functional complexity, displaying greater stability, broader lytic potential, and improved efficacy against bacterial pathogens (M. Iyer et al., 2021). Among these features, the broader lytic potential of jumbo phages is thought to be associated with the presence of tRNA and additional translation-related genes. These genes can reduce dependence on host cellular mechanisms and may contribute to a wider host range (Nazir et al., 2021). For example, phage ΦKZ encodes two putative RNA polymerases that enable infection independent of host RNAP activity (Ceyssens et al., 2014). Phage XacN1 contains 56 tRNA genes and exhibits a broader host range than other phages infecting Xanthomonas citri strains, which may be attributable to the presence of its multiple tRNAs (Yoshikawa et al., 2018).

In this study, we isolated a novel jumbo phage infecting A. hydrophila from aquatic environmental samples, designated it phage BMBAH1, and performed morphological observation, biological characteristic assessment, genome sequencing, and phylogenetic analysis. Whole-proteome phylogenetic analysis, gene-sharing network analysis, core protein clustering, and intergenomic similarity analysis consistently demonstrated that phage BMBAH1 belongs to the family Chimalliviridae while representing a distinct evolutionary lineage, supporting its designation as the founding member of a candidate novel viral genus, Southlakevirus. The present study expands the known phylogenetic diversity of A. hydrophila phages and provides a theoretical basis for further evaluation of BMBAH1 as a potential biocontrol agent in aquaculture and public health.

2. Materials and methods

2.1. Bacterial strains and culture conditions

All bacterial strains used in this study and their corresponding characteristics are Nlisted in Supplementary Table S1. A. hydrophila strains were kindly provided by the College of Fisheries, Huazhong Agricultural University (Wuhan, China). These strains had previously been isolated from liver and blood samples of diseased Pelodiscus sinensis (soft-shelled turtle) and identified as A. hydrophila by 16S rRNA gene sequencing. Other non-A. hydrophila strains used in this study were maintained in our laboratory. A. hydrophila strain CG was used for phage isolation and propagation, while the remaining strains were used for host range determination. All strains were cultured in Luria-Bertani (LB) broth at 37 °C.

2.2. Phage isolation and purification

To isolate phages infecting A. hydrophila, the lake water samples were collected from Nanhu Lake, Wuhan, Hubei Province, China. Subsequently, the water samples were centrifuged at 10,000 × g for 10 min to remove particulate matter and bacterial debris. The resulting supernatants were subsequently filtered through a 0.22-μm pore-size membrane filter (Millex syringe filter, Merck-Millipore) to obtain a relatively pure solution. The processed samples were stored at 4 °C until further experimentation. Phage isolation was performed as previously described with minor modifications (Hou et al., 2023). Briefly, aliquots of the filtered environmental samples were mixed with exponentially growing A. hydrophila cells (OD600 = 0.4) and incubated for 30 min to facilitate phage adsorption. The mixtures were then subjected to plaque detection using the double-layer agar method. Specifically, the mixtures were combined with molten soft LB agar and overlaid onto LB agar plates. After overnight incubation at 37 °C, individual plaques were identified and picked using sterile pipette tips. Single-plaque purification was repeated five times to obtain purified phage stocks.

2.3. Phage host range detection

The host range of the isolated phages was examined by a standard spot assay using 16 bacterial strains, including 11 A. hydrophila strains, 1 Aeromonas veronii strain, 2 Escherichia coli strains (E. coli BL21 and E. coli DH5α) (Xin et al., 2015), 1 Bacillus cereus ATCC 14579, and 1 Bacillus subtilis Bsn5 (Deng et al., 2011) (Table 1). Additional information for all bacterial strains used in this study is provided in Supplementary Table S1. Briefly, the high-titer phage suspension was first prepared by propagation using the host strain, followed by removal of bacterial debris through centrifugation and filtration using a 0.22-μm pore-size membrane filter (Millex syringe filter, Merck-Millipore). Phage particles were further concentrated by ultracentrifugation and resuspended in SM buffer (50 mM Tris–HCl, 100 mM NaCl, 8 mM MgSO4, 0.01% gelatin, pH 7.5). The phage titer was subsequently determined and adjusted to 10⁸ PFU/mL prior to use. For the spot assay, 100 μL of each test strain culture was spread on LB agar plates and allowed to dry at room temperature (RT) for 15 min. Then, 10 μL of a high-titer phage suspension (10⁸ PFU/mL) was spotted onto the surface of the solidified LB agar plates. The LB agar plates were then incubated at 37 °C overnight, and bacterial susceptibility to phage infection was determined based on the presence or absence of clear lytic zones. All experiments were performed in triplicate.

Table 1.

The host range of Aeromonas hydrophila phage BMBAH1.

Strain Medium Lytic activity
A. hydrophila JX-1 LB +
A. hydrophila JG2 LB +
A. hydrophila JG3 LB +
A. hydrophila JG9 LB +
A. hydrophila ATCC 7966 LB -
A. hydrophila B3 LB -
A. hydrophila HG13 LB -
A. hydrophila HG13–22 LB -
A. hydrophila CG LB +
A. hydrophila A13 LB -
A. hydrophila GG11 LB +
A. veronii ATCC 35624 LB -
E. coli BL21 LB -
E. coli DH5α LB -
B. cereus ATCC 14579 LB -
B. subtilis Bsn5 LB -
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Spot-like lysis of bacteria induced by phages: + indicates the production of a lytic zone, – indicates the absence of a lytic zone. Assays were performed with three independent biological replicates, with each measured in triplicate.

2.4. Phage thermal and pH stability assay

For thermal stability assay, 100 μL of phage suspension was incubated at eight different temperatures (4 °C, 28 °C, 37 °C, 42 °C, 50 °C, 60 °C, 70 °C and 80 °C) for 1 h. After incubation, the samples were serially diluted, and phage titers were determined using the double-layer agar method. For pH stability assay, phage suspensions were incubated in three different buffer systems with pH values of 2.0–12.0 for 1 h. Specifically, citrate-phosphate buffer composed of citric acid and disodium hydrogen phosphate (Na₂HPO₄) was used for pH 2.0–5.0, phosphate buffered saline (PBS; containing NaCl, KCl, Na₂HPO₄, and KH₂PO₄) was used for pH 5.0–8.0, and glycine-NaOH buffer composed of glycine adjusted with NaOH was used for pH 8.0–12.0. Phage titers were determined by the double-layer agar method described above. All experiments were performed in triplicate, and the experimental data are presented as mean ± standard deviation (SD).

2.5. Determination of multiplicity of infection (MOI)

The optimal multiplicity of infection (MOI) was determined according to previously described methods (Li et al., 2024). Briefly, the host strain was cultured in LB broth until reaching the mid-exponential growth phase (OD₆₀₀ = 0.4) with a final concentration of 10⁸ CFU/mL. To test different MOIs (100, 10, 1, 0.1, 0.01, and 0.001), 100 μL of serially diluted phage lysate was mixed with 100 μL of the host bacterial culture, added with 800 μL of fresh LB broth to reach a final volume of 1 mL. The mixtures were incubated at 37 °C with shaking at 200 rpm for 5 h. After incubation, phage titers were determined using the double-layer agar method. The MOI with the highest phage titer was defined as the optimal MOI. All MOI conditions were tested in triplicate, and the experimental data are presented as mean ± SD.

2.6. Adsorption efficiency and one-step growth assay

The adsorption efficiency experiment of phage was determined according to a previously described method with minor modifications (Hou et al., 2023). Briefly, exponentially growing A. hydrophila CG was mixed with the phage suspension at an MOI of 0.1 and incubated at 37 °C. Samples were collected at 2, 4, 6, 8, 10, 15, and 20 min and centrifuged at 10,000 × g for 1 min to separate free (unadsorbed) phages from bacterial cells. The titers of unadsorbed free phages in the supernatants were subsequently determined using the double-layer agar method, and the initial phage titer without bacterial cells was also determined in parallel using the same method. The phage adsorption efficiency was calculated using the following equation: Adsorption efficiency (%) = (N0−Nt)/N0×100, where N0 represents the initial phage titer and Nt represents the titer of unadsorbed phages at each sampling time point. Each sample was tested in triplicate, and the experimental data are presented as mean ± SD.

A one-step growth curve experiment was conducted according to a previously described method (Wen et al., 2025). The host bacteria were cultured to the exponential phase, after which 5 mL of phage suspension was mixed with 5 mL of the bacterial culture to obtain an MOI of 0.1 and incubated at 37 °C for 10 min to allow phage adsorption. Afterwards, the mixture was centrifuged at 10,000 × g for 2 min, and the supernatant was discarded. The precipitate was resuspended in 10 mL of LB broth and subsequently incubated at 37 °C with shaking at 200 rpm. During incubation, 100 μL samples were collected at 10 min intervals, serially diluted, and plated using the double-layer agar method for phage enumeration. The latent period was defined as the time from the completion of adsorption (t = 0) to the first significant increase in extracellular phage titer. Burst size was calculated as the ratio of the phage titer at the end of the burst period relative to the phage titer during the latent period (Chen et al., 2023). Each sample was performed in triplicate, and the experimental data are presented as mean ± SD.

2.7. Transmission electron microscopy (TEM) of phage BMBAH1

Approximately 5 μL of phage suspension (1 × 1010 PFU/mL) was dripped onto 200-mesh steel grids with 2–10 nm carbon layers and adsorbed for 10 min. Then, the samples were subjected to negative staining with 2% uranyl acetate for 90 s. The grid was washed with ultrapure water and dried on filter paper for 3 h. The images of phages were captured using an H-7650 transmission electron microscope at 100 kV in the Public Laboratory of Electron Microscopy, Huazhong Agricultural University (Wuhan, China). A total of eight independent phage particles (n = 8) were selected and measured for morphological analysis, and the experimental data are presented as mean ± SD.

2.8. In vitro antibacterial assay of phages

Phage suspension was diluted to obtain different multiplicities of infection (MOIs = 10, 1, 0.1, and 0.01). For each MOI, 100 μL of phage suspension was mixed with 100 μL of exponentially growing host bacterial culture (1 × 106 CFU/mL) (at 1:1 ratio v/v) and transferred into a 100-well microplate. Well containing 100 μL of LB medium and 100 μL of SM buffer served as the control. The 100-well plates were incubated in an automated microbial growth curve analyzer, and optical density at 600 nm (OD₆₀₀) was measured at 30-min intervals for 24 h. Bacterial growth and lysis curves were plotted with time as the x-axis and OD₆₀₀ values as the y-axis to analyze bacterial inhibitory dynamics by the phage at different MOIs. The assay of each sample was performed in triplicate, and the experimental data are presented as mean ± SD.

2.9. Phage DNA extraction and whole genome sequencing

Phage genomic DNA was extracted following a previously described method (Wang et al., 2024). Specifically, the phages were first treated with DNase I (2000 U/mL, 10 μL) and RNase A (10 mg/mL, 5 μL) (TaKaRa, Japan) at 37 °C for 1 h to prevent bacterial nucleic acid interference. Subsequently, the treated phages were added with sodium dodecyl sulfate (20% SDS, 20 μL) and proteinase K (20 mg/mL, 5 μL) and incubated at 37 °C for 2 h to lyse phage and degrade proteins to obtain phage DNA. Finally, the resulting phage DNA was purified by phenol-chloroform extraction and precipitated with anhydrous alcohol (Sambrook and Russell, 2006). Whole-genome sequencing (WGS) was conducted in paired-end mode (2 × 150 bp) using an Illumina NovaSeq 6000 sequencing system (Illumina). The quality of the raw phage sequencing reads was assessed using FastQC (v0.12.1) (Chen et al., 2018), and adapter sequences and low-quality bases were removed with Trimmomatic (v0.36) (Bolger et al., 2014). The obtained high-quality reads were de novo assembled using Unicycler v0.5.1 (Wick et al., 2017). PhageTerm (v1.0.12) was used to identify the genomic termini and DNA packaging strategy of the phage (Garneau et al., 2017). Open reading frames (ORFs) in the genome were predicted with the Rapid Annotation using Subsystem Technology (RAST) (Overbeek et al., 2014), and tRNA genes were identified using tRNAscan-SE (v2.0) (Chan and Lowe, 2019).

2.10. Bioinformatics analysis

The complete genome of phage BMBAH1 was compared with the genome sequences of characterized bacteriophages in the NCBI GenBank database using BLASTn for homology search (Camacho et al., 2009). To evaluate potential virulence genes, the complete phage genome sequences were aligned against the Virulence Factor Database (VFDB) (Chen et al., 2005). Predicted protein sequences were also aligned against the NCBI RefSeq non-redundant (nr) protein database using BLASTp (v2.17.0) and the Prokaryotic Virus Remote Homologous Groups (PHROG) database for homology-based annotation of predicted genes (Terzian et al., 2021), and the screening threshold parameters were set as follows: E-value ≤ 1 × 10–5, identity ≥ 40%, and coverage ≥ 60%. When multiple hits met these criteria, the best-supported annotation was selected based on sequence similarity, coverage, and functional relevance. Finally, a circular genome map of the phage was plotted using CGView software (Grant and Stothard, 2008). Preliminary taxonomic classification was performed using PhaBOX (Shang et al., 2023) and taxMyPhage (Millard et al., 2025). Intergenomic similarity between phage BMBAH1 and its closest relatives was calculated using VIRIDIC (Moraru et al., 2020). vConTACT3 was used to construct a gene-sharing network based on shared protein clusters to predict the genus-level taxonomic placement of phage BMBAH1 (Bolduc et al., 2025). VirClust was used to cluster viral genomes based on shared protein similarity (Moraru, 2023). Phylogenetic relationships and comparative genomic synteny among phage genomes were analyzed using the ViPTree server (Nishimura et al., 2017). Multiple sequence alignment of amino acid sequences was performed using MAFFT v7 (Katoh et al., 2019). The sequences of the major capsid protein (MCP), terminase large subunit (TerL), and DNA helicase in phage BMBAH1 and representative reference phages were aligned separately. Phylogenetic trees were constructed by maximum-likelihood method using IQ-TREE with 1000 ultrafast bootstrap replicates (Minh et al., 2020). Phylogenetic trees were visualized using the Interactive Tree of Life (iTOL) (Letunic and Bork, 2024).

2.11. Statistical analysis

Statistical analysis and figure plotting were performed using GraphPad Prism 10.0 software. For in vitro antibacterial assays, one-way ANOVA followed by Tukey’s post hoc multiple comparison test was performed, and differences were considered statistically significant at p < 0.05.

3. Results

3.1. Isolation of phage BMBAH1

Phage BMBAH1 was isolated from a lake water sample using A. hydrophila CG as the host strain. In the spot assay, phage BMBAH1 produced clear and well-defined lytic zones on double-layer agar plates, indicating successful lytic infection (Fig. 1A). TEM examination showed that phage BMBAH1 possessed a long tail (210 ± 2.0 nm in length) and an icosahedral head (51 ± 2.0 nm in diameter) (Fig. 1B, C).

Fig. 1.

Fig. 1

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Morphology of phage BMBAH1. (A) Spot assay in a 9-cm-diameter Petri dish. (B, C) TEM images of phage BMBAH1. The scale bar = 100 nm (bottom).

3.2. Host range determination of BMBAH1

To determine the host range of phage BMBAH1, a total of sixteen bacterial strains were tested in this study, including eleven A. hydrophila strains, as well as three Gram-negative bacteria (E. coli BL21, E. coli DH5α and A. veronii ATCC 35624) and two Gram-positive bacteria (B. cereus ATCC 14579 and B. subtilis Bsn5). The spot assay results showed that six of the eleven A. hydrophila strains formed clear lytic zones on double-layer agar plates, indicating their susceptibility to phage BMBAH1. No lytic zones were observed against any of the non-A. hydrophila strains (Table 1).

3.3. Thermal stability test

The thermal stability of phage BMBAH1 was evaluated after 1-hour incubation at different temperatures (Fig. 2A). The results showed that, after incubation below 60 °C, the phage titer exhibited good stability, remaining above 108 PFU/mL. However, when the incubation temperature increased to 70 °C, the titer of phage BMBAH1 decreased by approximately two orders of magnitude. After treatment at 80 °C, no viable phages were detected. These results indicate that phage BMBAH1 exhibits considerable thermal tolerance up to 60 °C, while exposure to higher temperatures reduces its viability.

Fig. 2.

Fig. 2

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Biological characteristics of phage BMBAH1. (A) Thermal stability. (B) pH stability. (C) Multiplicity of infection. (D) Adsorption assay. (E) One-step growth curve.

3.4. pH stability test

The pH stability assay showed that, after 1-hour incubation at pH 5.0–11.0, high titers of phage BMBAH1 were observed, remaining above 10⁹ PFU/mL (Fig. 2B). At pH 4.0, the phage titer decreased by approximately one order of magnitude, but it remained above 10⁸ PFU/mL. At pH 12.0, the phage titer dropped to below 107 PFU/mL. Under extremely acidic conditions (pH 2.0), the phage was completely inactivated, indicating irreversible denaturation of its capsid in such an acidic environment.

3.5. Optimal multiplicity of infection

To determine the optimal multiplicity of infection (MOI) for phage BMBAH1, the host bacteria were infected with phage BMBAH1 at six different MOIs (100, 10, 1, 0.1, 0.01, and 0.001) for 5 h, and then phage titers were measured. The results showed that an MOI of 0.1 produced the highest phage titer (2.25 × 10¹⁰ PFU/mL), which was higher than those observed under the other MOI conditions (Fig. 2C). Therefore, the optimal MOI was determined as 0.1.

3.6. Adsorption efficiency and one-step growth curve

The adsorption assay showed that phage BMBAH1 was rapidly adsorbed to A. hydrophila CG. Approximately 54.3% of phage particles were adsorbed within 2 min, with adsorption efficiency reaching 70.3% at 6 min and exceeding 83.4% after 10 min. It then remained at approximately 84–86%, indicating that most phage particles had completed adsorption within 10–15 min (Fig. 2D).

To further examine typical biological characteristics of phage BMBAH1 (such as the latent period and lytic capacity), a one-step growth experiment was conducted at an MOI of 0.1. The results showed that phage BMBAH1 had a latent period of approximately 30 min and a lytic period of about 80 min. During the stable phase, the titer of phage BMBAH1 reached 108 PFU/mL, and its burst size was approximately 212 ± 8 PFU/cell (Fig. 2E).

3.7. Phage in vitro lytic test

To evaluate the ability of the phage to lyse A. hydrophila, we investigated the long-term inhibitory effects of the phage on the isolated host strain during a 24-h co-culture period at four different MOIs (0.01, 0.1, 1, and 10). The results showed that phage BMBAH1 exhibited pronounced antibacterial activity under all tested MOI conditions. During the early stage of co-culture (0–4 h), a transient increase in OD₆₀₀ values was observed in the phage-treated bacterial groups, which may be attributed to the relatively low initial phage abundance allowed short-term bacterial growth. However, after 4 h, bacterial OD₆₀₀ values in the phage-treated groups began to decrease compared with those in the control group, indicating phage-mediated lytic effects. Subsequently, the inhibitory effect of BMBAH1 persisted for up to 16 h. Nevertheless, a slight increase in bacterial growth was observed again after 16 h, which may be associated with the emergence of phage-resistant or phage-tolerant bacterial subpopulations (Oechslin, 2018). Overall, higher MOI values generally produced stronger inhibition of bacterial growth, suggesting MOI-dependent antibacterial activity of phage BMBAH1 (Fig. 3A, B).

Fig. 3.

Fig. 3

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In vitro inhibitory effect of phage BMBAH1 against A. hydrophila. (A) Growth curves of A. hydrophila CG co-cultured with phage BMBAH1 at different MOIs. The group without phage BMBAH1 served as the control, while the groups co-cultured with phage BMBAH1 at different MOIs served as the treatment groups. (B) Final OD600 values of A. hydrophila CG at 24 h after phage addition at different MOIs (corresponding to panel A). Group differences were statistically assessed using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. **** p < 0.0001.

3.8. Genome characterization and annotation

The complete genome of phage BMBAH1 was 237,469 bp in length and was therefore classified as a jumbo phage. Its genome consisted of linear double-stranded DNA (dsDNA) with a GC content of 44.3%. BLASTn analysis revealed only limited nucleotide alignments between phage BMBAH1 and all currently available phage genomes in the NCBI database, with query coverage values below 6%. These results indicate that phage BMBAH1 is highly divergent from previously reported phages at the genomic level. A total of 241 ORFs were predicted using RAST and subsequently annotated using NCBI BLASTp. Among them, 46 ORFs were annotated as proteins with known functions, whereas 195 ORFs were annotated as hypothetical proteins. In addition, 4 tRNAs were identified within the genome. Based on predicted protein functions, all of the proteins of phage BMBAH1 were classified into six functional modules, including DNA replication, regulation and nucleotide metabolism, phage packaging, phage structure, host lysis, auxiliary metabolism and other functions (Fig. 4). No putative toxins and antibiotic-resistance genes were detected.

Fig. 4.

Fig. 4

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Genome map of phage BMBAH1. Circles display (from the inside to the outside): (1) The inner circle indicates the full length of the phage BMBAH1 genome. (2) GC skew plots of (G – C)/(G + C) are shown in green and purple. (3) G + C % content. (4) The gene coding regions transcribed in the clockwise or the counterclockwise directions. Different colors represent various types of functional modules in the genome. Additionally, key functional genes within each module are labeled along the map for clear identification.

Specifically, the DNA replication, regulation, and nucleotide metabolism module included genes encoding several representative proteins, including DNA polymerase (ORF 61), DnaB-like replicative helicase (ORF 65), DNA ligase (ORF 38), and DNA-directed RNA polymerase subunits (ORFs 101, 119, and 185). The phage packaging module contained genes encoding a portal protein (ORF 57), DNA terminase (ORF 99), and terminase large subunit (ORF 160), all of which are likely involved in DNA processing and genome packaging. The structural module included genes encoding several representative proteins, including the major head protein (ORF 63), capsid and scaffold protein (ORF 80), tail protein (ORFs 46, 47, and 48), and tail sheath protein (ORF 164). The host lysis module mainly included an endolysin (ORF 183) and a transglycosylase (ORF 42). In addition, several auxiliary metabolism-related proteins, including chaperonin GroEL (ORF 214) and several acetyltransferase-related proteins, were also identified (Table S2). The other functions module comprised genes that were not classified into the above functional categories, including a tubulin-like PhuZ protein (ORF 130), which may participate in the intracellular positioning of phage components during infection.

3.9. Phage BMBAH1 represents a candidate novel viral genus

To determine the taxonomic position of phage BMBAH1, we first performed a preliminary classification prediction using PhaBOX and taxMyPhage. The results showed that phage BMBAH1 could not be assigned to any currently established viral genus, suggesting that it may represent a previously undescribed taxonomic unit.

Under current International Committee on Taxonomy of Viruses (ICTV) phage taxonomy frameworks, viral classification is generally supported by multiple complementary analyses, including proteome-based phylogeny, gene-sharing network approaches, whole-genome similarity analysis, and conserved marker gene phylogenies (Turner et al., 2021). Based on this framework, we constructed a whole-proteome-based phylogenetic tree using ViPTree to elucidate the overall phylogenetic position of phage BMBAH1. The phylogenetic tree showed that phage BMBAH1 exhibited a relatively close phylogenetic relationship with several previously reported Chimalliviridae phages, including Aeromonas phages D3, D6, and LAh10. These phages formed a distinct phylogenetic clade that was clearly separated from other lineages (Fig. 5A). Meanwhile, a gene-sharing network constructed with vConTACT3 produced a similar pattern. In the resulting network, phage BMBAH1 clustered together with 20 other phages, including Aeromonas phages D3, D6, and LAh10, forming a distinct viral cluster (VC) (Fig. 5B). All members of this cluster belong to the family Chimalliviridae. Consistent with the ViPTree analysis, these results indicate that phage BMBAH1 shares a relatively close evolutionary relationship with these phages.

Fig. 5.

Fig. 5

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(A) Proteomic trees constructed based on proteomic alignment using ViPTree. The viral proteomic tree includes phage BMBAH1 and some phages against other hosts. Phage BMBAH1 is labeled with a red line. (B) Gene-sharing network generated by vConTACT3. Each circle (node) represents a genome, and connecting lines (edges) represent similarity between genomes based on shared protein clusters. Phage BMBAH1 clustered with 20 other phages to form a distinct viral cluster. (C) Shared-gene analysis of phage BMBAH1 and related phages using VirClust. Phage BMBAH1 shares a high proportion of homologous genes with phages assigned to the genera Ludhianavirus and Ferozepurvirus within the family Chimalliviridae. Taxonomic assignments of reference phages are indicated in the figure. Phage BMBAH1 is labeled in red. (D) Phylogenetic analysis of phage BMBAH1 based on conserved proteins. Phylogenetic tree constructed based on the amino acid sequences of the major capsid protein (MCP) via the maximum-likelihood method. The phage BMBAH1 is highlighted in red to emphasize its evolutionary placement. Bootstrap values ≥ 50% are indicated at the corresponding nodes.

To further investigate the genomic relatedness and gene-sharing characteristics among the above phages, VirClust analysis was performed. The results showed that phage BMBAH1 shared 153 homologous genes with members of the family Chimalliviridae, including phages D3, D6, and LAh10, accounting for >60% of the total predicted ORFs within these genomes (Fig. 5C). In contrast, no shared genes were detected between phage BMBAH1 and members of the family Autotranscriptaviridae. Consistent results were also obtained from comparative genomic synteny analysis, which showed that multiple homologous regions were shared between BMBAH1 and the three reference phages (Fig. S1).

In addition, to further confirm the phylogenetic relationships inferred from shared orthologous genes, we selected three widely used conserved proteins in phages, including the major capsid protein (MCP) (Wang et al., 2024), the terminase large subunit (TerL) (Morozova et al., 2023), and DNA helicase (Wong et al., 2013), and constructed phylogenetic trees based on their amino acid sequences using the maximum-likelihood method. The phylogenetic analysis of MCP showed that phage BMBAH1 clustered with phages D3, D6, and LAh10, forming an independent evolutionary branch clearly separated from other phages (Fig. 5D). The phylogenetic trees of TerL and DNA helicase exhibited a highly similar topology to that of MCP (Fig. S2A, B). Taken together, these results indicate that phage BMBAH1 can be assigned to the family Chimalliviridae.

The genus-level taxonomic position of phage BMBAH1 was then evaluated using VIRIDIC. We collected a total of 100 publicly available complete A. hydrophila phage genome sequences from the NCBI database, including representative phages closely related to phage BMBAH1. VIRIDIC-based intergenomic similarity analysis showed that phage BMBAH1 exhibited low genomic similarity to all analyzed phages. The closest related phages were Aeromonas phage D3 (30.7%), Aeromonas phage LAh10 (30.4%), and Aeromonas phage D6 (30.3%) (Fig. 6). According to the guidelines of the ICTV, phages are considered to belong to the same genus when they share > 70% intergenomic similarity (Turner et al., 2021). Therefore, we propose phage BMBAH1 represents a candidate novel viral genus, designated Southlakevirus.

Fig. 6.

Fig. 6

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VIRIDIC-based heatmap of phage BMBAH1 and its closely related phages. Phage BMBAH1 is indicated in red. The right half shows similarity values between phage genomes. The left half indicates the aligned genome fraction and genome length ratio for a selected pair of phage genomes.

4. Discussion

A. hydrophila is an important pathogen in aquaculture, and the increasing antibiotic resistance poses a serious threat to the aquaculture industry (Barger et al., 2021). Phage therapy has been considered a promising alternative to antibiotics (Dong et al., 2021; Nithin et al., 2021). In this study, we isolated and characterized a novel A. hydrophila phage, designated phage BMBAH1. Transmission electron microscopy revealed that phage BMBAH1 possessed an icosahedral head (51 ± 2.0 nm in diameter) and a long tail (210 ± 2.0 nm in length). This phage was classified as a jumbo phage due to its large genome size (237,469 bp). Previous studies have generally suggested that jumbo phages tend to exhibit larger capsid dimensions than non-jumbo phages (Harding et al., 2023). However, in this study, phage BMBAH1 exhibited a relatively small capsid size despite its large genome. Similar compact head morphology has also been reported in other jumbo Aeromonas phages, such as ZPAH34 (234,546 bp, 53.4 ± 1.7 nm) (Hou et al., 2023) and Z90 (234,057 bp; 53 ± 2 nm) (Zhang et al., 2025). By comparison, jumbo Aeromonas phage P19 (228,239 bp) exhibited a larger capsid measuring 82.53 ± 2.3 nm (Ansari and Nagar, 2026). These comparisons indicate that capsid size in jumbo phages is not strictly correlated with genome length. Instead, the capsid size may be affected by multiple factors such as capsid assembly constraints, DNA packaging density (Roos et al., 2007), and structural protein composition (Yuan and Gao, 2017). In addition, technical factors associated with negative-stain transmission electron microscopy may also contribute to minor variations in measured particle dimensions. For example, sample preparation steps such as dehydration and air-drying may induce minor particle flattening or shrinkage, leading to slight underestimation of virion size (Mast and Demeestere, 2009).

As natural bactericidal microorganisms, phages are affected by multiple physicochemical environmental factors, and thus their stability under different environmental stresses is a key indicator for evaluating their potential as food biocontrol agents (Tomat et al., 2014). The biological characteristics of phage BMBAH1 further highlight its application potential as a therapeutic phage. First, phage BMBAH1 exhibited high biological activity over a wide temperature range. Its titer could remain as high as approximately 108 PFU/mL within the range of ≤ 60 °C, and it displayed relatively high infectivity even after exposure to 70 °C. Compared with previously reported jumbo Aeromonas phages, phage BMBAH1 displayed substantially greater thermal stability. For instance, phage ZPAH34 declined to approximately 10³ PFU/mL after incubation at 60 °C and 70 °C for 1 h (Hou et al., 2023). Similarly, phage Z90 exhibited a marked reduction in infectivity at 50 °C and completely lost activity at 60–80 °C (Zhang et al., 2025). The jumbo phage P19 also showed limited tolerance to elevated temperatures, with reductions of approximately 3.5-log and 4-log in phage titer after 20 min of exposure at 70 °C and 80 °C, respectively (Ansari and Nagar, 2026). Collectively, these findings indicate that phage BMBAH1 shows comparatively higher thermal stability than other reported jumbo Aeromonas phages, highlighting its potential environmental adaptability and suitability for phage-based biocontrol applications.

In addition to its thermal tolerance, phage BMBAH1 also remained active across a broad pH range. High phage titers were maintained from pH 4.0 to 11.0, covering moderately acidic and alkaline conditions. Similar pH tolerance profiles have also been reported in other jumbo Aeromonas phages, including Z90, ZPAH34, and P19, which generally remained stable across a wide pH range but showed substantial loss of viability under extreme pH stress (Ansari and Nagar, 2026; Hou et al., 2023; Zhang et al., 2025). The comparable pH stability observed among these Aeromonas phages suggests that tolerance to pH fluctuations may represent a common characteristic of this phage group. Such broad pH adaptability may facilitate the persistence and biological activity of phage BMBAH1 under diverse environmental conditions, supporting its potential application in phage-based biocontrol strategies.

Furthermore, the replication characteristics of BMBAH1 also differed in several respects from those of previously reported jumbo Aeromonas phages. Its one-step growth curve showed a latent period of approximately 30 min and a burst size of about 212 ± 8 PFU/cell. Compared with other reported jumbo Aeromonas phages, phage BMBAH1 displayed a longer latent period than ZPAH34 (20 min) and Z90 (20 min) but a larger burst size than ZPAH34 (79 PFU/cell) and P19 (50 PFU/cell), and was comparable to Z90 (270 PFU/cell) (Ansari and Nagar, 2026; Hou et al., 2023; Zhang et al., 2025). These findings indicate that BMBAH1 has strong replication and progeny production capacity. Although its latent period was slightly longer than that of some reported jumbo Aeromonas phages, BMBAH1 exhibited a comparatively high burst size. The relatively high burst size may enhance antibacterial efficacy through substantial progeny release during each infection cycle, thereby facilitating efficient phage amplification and bacterial clearance.

Moreover, lysis curves demonstrated that phage BMBAH1 continuously suppressed host bacterial growth, indicating its advantage in long-term control of bacterial proliferation and its potential as a candidate biocontrol or therapeutic agent (Park et al., 2012). However, partial bacterial regrowth was observed at later stages of infection under lower multiplicity of infection (MOI) conditions. This phenomenon may reflect the dynamic coevolutionary interactions between phages and bacterial populations and could result from the emergence of phage-resistant or phage-tolerant bacterial subpopulations. Previous studies have reported bacterial regrowth after initial lysis by phages during phage therapy. This phenomenon is mainly caused by modifications of phage receptors, activation of the CRISPR-Cas system, and other antiviral defense systems in bacteria (Oechslin, 2018). Such regrowth may compromise the long-term efficacy and stability of phage-based antibacterial strategies in complex environments. Therefore, optimization strategies such as phage cocktail formulations or the combined application of phages with other antibacterial agents may be required to suppress bacterial regrowth and enhance the long-term stability and durability of antibacterial efficacy in practical biocontrol applications (Malik et al., 2017).

A notable feature of the BMBAH1 genome is the exceptionally high proportion of hypothetical proteins. >80% of the predicted ORFs lacked functional annotation or detectable homologs in current public databases, indicating substantial genomic novelty and genetic divergence. This characteristic is commonly observed among jumbo phages, which frequently encode lineage-specific genes and previously unexplored functional proteins (Mesyanzhinov et al., 2002).

The functionally annotated genes in BMBAH1 were primarily associated with DNA replication, transcription, and nucleotide metabolism, including genes encoding DNA polymerase, helicase, ligase, and multiple DNA-directed RNA polymerase subunits. Jumbo phages are known to encode extensive replication and transcriptional machinery that can partially reduce their dependence on host cellular systems during infection (Nazir et al., 2021). The identification of multiple RNA polymerase subunits in BMBAH1 suggests that this phage likely possesses a relatively autonomous transcriptional system, similar to that reported in phiKZ-like jumbo phages (Zhang et al., 2025). Such transcriptional autonomy may support efficient intracellular replication and enable infection under diverse physiological conditions.

Genes associated with host lysis were also identified, including those encoding endolysins and transglycosylases. Endolysins and transglycosylases facilitate the degradation of bacterial peptidoglycan, thereby promoting efficient host cell lysis and progeny release (Briers, 2019; Hong et al., 2025). Interestingly, although endolysin-encoding genes were identified in the phage BMBAH1 genome, no holin gene was detected. This may be attributable to the high sequence diversity and limited conservation of holin proteins, which makes their identification through sequence-based annotation challenging. In addition, several auxiliary metabolism-related proteins, including chaperonin GroEL and acetyltransferase-related proteins, were identified. These proteins may contribute to protein folding, stress adaptation, and modulation of phage-host interactions during infection (van Duijn et al., 2005). Importantly, no putative virulence factors or antibiotic-resistance genes were identified in the genome of phage BMBAH1, suggesting that phage BMBAH1 might present a relatively low biosafety risk, which further supports its potential as a therapeutic or biocontrol phage (Endersen and Coffey, 2020).

Interestingly, phage BMBAH1 also encoded a tubulin-like PhuZ protein, which is a characteristic component of nucleus-forming jumbo phages. Previous studies have demonstrated that PhuZ participates in the formation and positioning of nucleus-like compartments during phage infection, thereby spatially organizing viral DNA replication and transcription while protecting phage genomes from host defense systems (Chaikeeratisak et al., 2017; Kraemer et al., 2012). The presence of this protein suggests that phage BMBAH1 may utilize a sophisticated intracellular compartmentalization strategy resembling that of other nucleus-forming jumbo phages.

Future studies combining transcriptomics, proteomics, and functional genetics will be necessary to clarify the biological roles of the numerous uncharacterized genes in BMBAH1 and to further elucidate the molecular mechanisms underlying its infection strategy and host interactions.

5. Conclusion

In this study, we characterized BMBAH1, a novel jumbo phage infecting A. hydrophila. Biological characterization demonstrated that phage BMBAH1 possessed broad temperature and pH stability, strong replication capability with a large burst size, and sustained antibacterial activity against A. hydrophila, highlighting its potential as a therapeutic and biocontrol agent. Phage BMBAH1 possessed a large and complex genome containing numerous hypothetical proteins, highlighting the unexplored genetic diversity of this jumbo phage. Whole-genome comparisons, protein clustering, phylogenetic tree analyses, and intergenomic similarity analyses consistently demonstrated that phage BMBAH1 represents a candidate novel viral genus within the family Chimalliviridae. Given that the genomic diversity and evolutionary relationships of jumbo Aeromonas phages remain poorly characterized, BMBAH1 may represent a highly divergent lineage within Aeromonas-associated jumbo phages. The identification and characterization of phage BMBAH1, therefore, expand the current taxonomic framework of jumbo phages and provide additional insights into the evolution, diversity, and genomic complexity of Chimalliviridae-related phages.

Ethics declaration

The author(s) declare(s) that the study does not involve humans nor animal subjects.

Nucleotide sequence number

The complete genome sequence of phage BMBAH1 has been deposited in the GenBank database under accession number PX879978.

Declaration of competing interest

The final version was examined and approved by all authors, who also take joint responsibility for looking into and addressing any issues with the accuracy or integrity of the work.

CRediT authorship contribution statement

Shitian Huo: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Data curation, Conceptualization. Pengfei Zhu: Data curation. Jikai Xu: Methodology. Kai Wang: Writing – review & editing. Ming Sun: Writing – review & editing. Donghai Peng: Writing – review & editing, Resources, Project administration.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We gratefully acknowledge the College of Fisheries, Hubei Province, Huazhong Agricultural University for providing the strain used in this study.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2026.199813.

Appendix. Supplementary materials

mmc1.docx (496.5KB, docx)
mmc2.docx (28.4KB, docx)

Data availability

The data that support the findings of this study are available from the corresponding author on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.docx (496.5KB, docx)
mmc2.docx (28.4KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author on reasonable request.

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