Characterization and Genome Analyses of the Novel Phages P2 and vB_AhydM-H1 Targeting Aeromonas hydrophila

Abstract

Background:

The emergence of antibiotic-resistant Aeromonas hydrophila strains presents a global health and aquaculture challenge. Bacteriophages offer promise as an alternative to antibiotics for treating drug-resistant Aeromonas infections.

Methods:

Two new phages, P2 and vB_AhydM-H1, targeting pathogenic A. hydrophila were isolated from sewage water. Their morphology, growth characteristics, lytic activity, stability, and genomes were analyzed.

Results:

Phage P2, a member of genus Ahphunavirus, and vB_AhydM-H1, a novel member of genus Pahsextavirus, exhibited narrow host ranges, extended latent periods, and typical burst sizes. Both phages remained stable at 40°C for 1 h and within a pH range of 4 to 10 for 3 h. The genomes of P2 and vB_AhydM-H1 spanned 42,660 bp with 49 open reading frames (ORFs) and 52,614 bp with 72 ORFs, respectively. Proteomic (ViPTree) and phylogenetic (VICTOR) analyses confirmed that both phages aligned with their respective families. DeepTMHMM predictions suggested that P2 and vB_AhydM-H1 encode three and four ORFs with transmembrane domains, respectively.

Conclusions:

Safe for environmental and clinical use because of their lytic nature, and lack of virulence and resistance genes, these newly isolated phages expand the arsenal against antibiotic-resistant Aeromonas infections.

Introduction

Aeromonas hydrophila, a ubiquitous Gram-negative bacterium within the genus Aeromonas, embodies the “jack-of-all-trades” label in the One Health context. Its pervasive presence across diverse aquatic, environmental, and food ecosystems poses a significant public health challenge.¹ It reigns as a primary pathogen for fish, causing diseases such as hemorrhagic septicemia and furunculosis, while also inflicting a variety of intestinal and extraintestinal infections in humans. Its adeptness at biofilm formation, virulence factor production, and harboring of integrons coupled with multidrug resistance strains presents a formidable obstacle in disease prevention and treatment.² A. hydrophila has been linked to documented foodborne outbreaks and implicated in large-scale aquaculture catastrophes worldwide, inflicting substantial economic losses.³ Notably, India faces a concerning rise in antibiotic-resistant Aeromonas strains from both clinical sources and environmental reservoirs.^4,5 While chemical antimicrobials are frequently employed to control foodborne pathogens in pre- and postharvest stages of food and aquaculture production, their widespread application across human, veterinary, and aquaculture settings has fueled the global emergence and dissemination of antimicrobial-resistant human pathogens, leading to hefty human and economic costs.⁶

Antibiotic resistance necessitates exploring alternative antibacterial solutions. Bacteriophages, naturally occurring bacterial viruses, offer promise.⁷ They selectively target specific bacteria, thereby minimizing harm to beneficial microbes. Additionally, they self-regulate, replicating only in the presence of their host, and pose minimal safety risks.⁸ DNA sequencing advancements streamline phage analysis, ensuring safety and novelty for biocontrol. Whole genome sequencing helps identify phages lacking antibiotic resistance or virulence genes.⁹ Phylogenetic analysis aids in phage classification, thus facilitating discovery of novel isolates.^10,11 Diverse phage genomes enable identification of potentially beneficial genes and improve understanding of phage–bacteria interactions.¹² This knowledge paves the way for utilizing novel phage proteins and phages themselves in various applications, including medicine and biotechnologies.¹³ Phages’ targeted antibacterial properties and ability to minimize adverse effects from bacteria broaden their potential in diverse fields such as aquaculture, agriculture, food safety, and human medicine.¹⁴

Despite documented A. hydrophila infections,^15,16 limited research exists on well-characterized bacteriophages targeting this bacterium. Well-characterized phage collections are critical for effective phage therapy against A. hydrophila. These repositories provide a backup of therapeutic phages and enable the development of broader-spectrum phage cocktails. This study attempts to address this gap by isolating two lytic phages from sewage targeting virulent and antibiotic-resistant A. hydrophila strains. We characterized these phages for their host range, morphological features, growth kinetics, temperature, and pH stability, as well as their genomes for taxonomic classification and biocontrol safety evaluation. This comprehensive characterization paves the way for robust A. hydrophila phage repositories for future applications.

Material and Methods

Bacterial strains and culture conditions

A. hydrophila strains, CECT 839^T and A331 (source, antibiotic resistance, and virulence details in Supplementary Table S1), were grown in tryptic soya broth (TSB) and on tryptic soya agar (TSA) (HiMedia, India) at 30°C for phage enrichment and isolation.

Sample collection, phage enrichment and isolation

Sewage samples from Mumbai and Pune, India, were processed to isolate phages according to Kropinski et al.¹⁷ Samples were collected in sterile containers and clarified by centrifugation at 10,000 g for 10 min. The supernatant was then filtered using a 0.22 µm sterile syringe filter (Sartorius, Germany) to remove any remaining debris. The filtrate was mixed with an equal volume of double-strength TSB, supplemented with 1 mM CaCl₂, and overnight-grown cultures of A. hydrophila (1% v/v) for phage enrichment. After incubation at 30°C with gentle shaking, the enrichment culture was centrifuged at 10,000 g for 10 min. Supernatants showing bacterial lysis were used as the source of phages in plaque assays, performed by double agar overlay. In this assay, 100 µL of A. hydrophila culture was mixed with 100 µL of appropriately diluted phage lysate and premolten 0.5% agar, supplemented with 1 mM CaCl₂, and overlaid on TSA. Plates were then incubated at 30°C, and single plaques were picked and purified through three rounds of plaque assay. Following purification, phages were harvested from agar overlay plates containing semiconfluent host lysis by adding 5 mL of SM buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 8 mM MgSO4, 0.01% gelatin) to achieve high-titer phage stocks (∼ 10¹⁰ PFU/mL) and stored at 4°C.

High-titer phage stocks were prepared using standard methods.¹⁸ Ice-cold 30% (w/v) PEG 8000 and 3 M NaCl were added to the phage lysate at final concentrations of 10% (w/v) and 0.5 M, respectively. After overnight incubation at 6°C, the mixture was centrifuged at 10,000 g for 20 min at 4°C. Supernatant was decanted, and SM buffer was added. Following centrifugation to remove bacterial debris, chloroform treatment removed residual PEG. The aqueous phase was collected, designated as phage stock, and stored at 4°C. Phage titer was determined, and 20% (v/v) sterile glycerol added before storage at −80°C.

Host range

The host range of P2 and vB_AhydM-H1 phages was determined using a spot test, in triplicate, against various Aeromonas spp. and other bacteria (Supplementary Table S2).¹⁹ Briefly, 10 μL of high-titer phage stock (10¹⁰ PFU/mL) was serially diluted, spotted onto bacterial lawns, and incubated overnight at 30°C. The zone of clearance was observed to confirm the host lysis.

Phage morphology

Transmission electron microscopy (TEM) analysis revealed the morphological details of phages P2 and vB_AhydM-H1.²⁰ Briefly, high-titer lysates (>10¹⁰ PFU/mL) were negatively stained with 2% phosphotungstic acid on carbon-coated copper grids and visualized under a JEM-1400 Plus TEM (JEOL, Japan) at 120 kV at the Electron Microscope Facility, ACTREC, Navi Mumbai. Phage dimensions, measured using ImageJ v1.52a software,²¹ aided in taxonomic classification following ICTV guidelines (http://www.ictvonline.org/) and Novik et al.²²

One-step growth curve analysis

Adsorption rate, latent period, and burst size were determined for phages P2 and vB_AhydM-H1 by performing the one-step growth curve experiment with slight modifications.²³ Exponentially grown A. hydrophila cultures (∼10⁸ CFU/mL) were infected with phages (∼10⁶ PFU/mL) at an MOI of 0.01. Following adsorption for 10 min, unbound phages were removed. Phage titers were measured every 10 min for 3 h using double-layer agar plate method.¹⁷ Latent period and burst size were calculated from one-step growth curves (phage count vs. time) generated in triplicate experiments.

Phage stability assessment

Thermal and pH stability of phages P2 and vB_AhydM-H1 were evaluated based on methods from Fu et al.²⁴ Briefly, phage suspensions (9 × 10⁹ PFU/mL) were incubated at various temperatures (4–80°C) for 1 h, with 100 μL aliquots taken every 20 min for titer determination. For pH stability, 100 μL phage suspensions were added to 9.9 mL of autoclaved TSB, adjusted to pH 2–12 by addition of HCl and NaOH, and incubated at 30°C for 3 h. Titer was determined following serial dilution, using double-layer agar method. All experiments were performed in triplicate.

Phage DNA extraction and sequencing

Phage genomic DNA was extracted using a standard phenol–chloroform method.²⁵ Sequencing libraries were prepared with the NEBNext^® Ultra™ II FS DNA Library Prep Kit (Illumina) and quality controlled using TapeStation (Agilent) and Qubit (Thermo Fisher). Paired-end whole-genome sequencing (2 × 150 bp) was performed on an Illumina HiSeq 2500 system.

Genome Assembly and Analysis

The quality of sequencing data was assessed by visualization using FastQC v0.12.1.²⁶ Adapters were removed and low-quality sequences were trimmed using CutAdapt v1.10 and Trimmomatic v0.36.^27,28 The quality-filtered data were de novo assembled into complete genomes using SPAdes v3.7.1,²⁹ and phage termini were identified with PhageTerm tool.³⁰ Sequence and intergenomic similarities of P2 and vB_AhydM-H1 genomes to other phages (Supplementary Table S3) were assessed using nucleotide BLAST (BLASTn) against NCBI database and Virus Intergenomic Distance Calculator (VIRIDIC) tool (>70% and >95% similarity cutoffs for genus and species, respectively),³¹ respectively. Virus Classification and Tree Building Online Resource (VICTOR) analysis with GBDP (100 pseudo-bootstrap replicates) and OPTSIL³² methods determined their phylogenetic relationships for species, genus, and family demarcation. FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/fitree/). visualized the phylogenetic tree, while Viral Proteomic Tree (ViPTree) server 1.938 analyzed P2 and vB_AhydM-H1 relationships with known phages.³³ Rectangular genome comparison diagrams were generated for closely related phages (ViPTree score SG > 0.1). Gene prediction used GeneMark Suite³⁴ with specific parameters.³⁵ Functional assignments were made using NCBI Conserved Domain Search, BLASTp, InterProScan, and HHpred, ARAGORN and tRNAscan-SE. ResFinder 4.1 identified potential virulence and antibiotic resistance genes. Phage Classification Tool Set (PHACTS),³⁶ PhageAI,³⁷ and DeepTMHMM³⁸ computationally assessed phage lifestyle, taxonomy, and protein topology, respectively.

Results

Phage isolation, host range, and morphology

After phage enrichment and spot test, one sewage water sample, each from Mumbai and Pune, showed lytic activity against A. hydrophila CECT 839^T and A. hydrophila A331, respectively. Further double-layer agar plate method resulted in isolation and purification of phages, P2 (host A. hydrophila CECT 839^T) (Supplementary Fig. S1A) and vB_AhydM-H1 (host A. hydrophila A331) showing clear plaques (Supplementary Fig. S1B). Both phages were tested for infectivity against a diverse panel of bacteria, including Aeromonas strains, representing various species, and other bacterial species. However, their host range proved to be highly specific, limited only to their original isolation hosts, with no observed infection of other Aeromonas or non-Aeromonas strains (Supplementary Table S2).

TEM micrographs showed P2 phage with an icosahedral head of diameter 54.4 ± 1.5 nm (mean ± SD) and short tail of length 10.4 ± 1.1 nm, characteristic of podovirus, using ImageJ software (Fig. 1A); whereas, vB_AhydM-H1 possessed an icosahedral head (61.1 ± 2.3 nm) with a contractile tail (133.3 ± 2.7 nm) and tail fibers, characteristics of myovirus (Fig. 1B).

FIG. 1.

TEM micrograph of A. hydrophila phages (A) P2 and (B) vB_AhydM-H1 stained with 2% phosphotungstic acid. Scale bars represent 50 nm. TEM, Transmission electron microscopy.

One-step growth curve analysis

During the early phase of one-step growth curve analysis, approximately 90% of both phages adsorbed to their respective host cells within a 10-min incubation period. The replication dynamics revealed a latent period of ∼90 min for phage P2 (Fig. 2A) and ∼120 min for vB_AhydM-H1 (Fig. 2B), with both exhibiting a similar 90-min rise period. Burst size, calculated as the ratio of phage titer at the plateau phase to the latent phase, was 34 ± 7 PFU/cell for P2 and 179 ± 12 PFU/cell for vB_AhydM-H1 (mean ± SD).

FIG. 2.

One-step growth curve of A. hydrophila phages (A) P2 and (B) vB_AhydM-H1 at a multiplicity of infection value of 0.01. Data points represent the mean± SD of PFU/mL at different intervals during the experiment time. The results are the average of three replications with standard deviation as vertical lines.

Phage stability analysis

Both phages remained stable at 4°C, 30°C, and 40°C for 60 min (Fig. 3A and 3B). However, their titers decreased significantly after exposure to temperature greater than 50°C for 60 min. Incubation at 70°C and 80°C resulted in more than 3 log-cycle reductions in phage titers after 60 min. Both phages were stable in pH range from 6 to 10 with significant reduction in viability below pH 4 (Fig. 4).

FIG. 3.

Temperature stability of A. hydrophila phages (A) P2 and (B) vB_AhydM-H1 at different temperature range from 4°C to 80°C.

FIG. 4.

The pH stability of A. hydrophila phages P2 and vB_AhydM-H1 at pH range from 2 to 12.

Genome sequencing, annotation, and bioinformatics analysis

The QC filtered PE sequencing reads of P2 and vB_AhydM-H1 phages were de-novo assembled into single contigs, measuring 42,660 bp (410x coverage, 58.8% guanine-cytosine (GC) content) and 52,614 bp (215x coverage, 51.8% GC content), respectively (Table 1, Supplementary Table S4 and S5). PhageTerm analysis revealed that both phage genomes are circularly permuted with randomly located 5′ and 3′ termini. Using the GeneMark Suite, a total of 49 and 72 slightly overlapping ORFs were predicted on the plus strand of P2 and vB_AhydM-H1 phage genomes, respectively. Predicted ORF lengths ranged from 183 to 3,759 bp for P2 and 120 to 3,483 bp for vB_AhydM-H1 phages (Supplementary Table S4 and S5). The complete genome sequences of phages P2 and vB_AhydM-H1 have been deposited in the GenBank database under the accession numbers OP797796 and OR795024, respectively (Table 1).

Table 1.

Overview of the Phages

	P2	vB_AhydM-H1
GenBank Accession Number	OP797796	OR795024
Class	Caudoviricetes	Caudoviricetes
Family	Autographiviridae	Chaseviridae
Genus	Ahphunavirus	Pahsextavirus
Genome size (GC%)	42660 bp (58.8%)	52614 bp (51.8%)
Shape (circular/linear)	Linear	Linear
No. of ORFs	49	72
No. of promoters	75	108
No. of tRNAs	0	1 (tRNA-Gly-TCC)
Temperate lifestyle genes	No	No
No. of rho-independent transcription terminators	7 = 1 (F) + 6 (R)	15 = 8 (F) + 7 (R)
No. of antibiotic resistance genes	None	None
No. of virulence genes	None	None
Nearest neighbors based on BLASTn analysis—Name (% Identity, Accession number)	CF7 (97.4%, NC_047865.1) Ahp1 (96.1%, NC_047866) AHPMCC7 (95.9%, ON365567.2)	BUCT696 (91.6%, NC_079186.1) PVN04 (84.1%, MW380984.1) pAh6-C (77.0%, NC_025459.1)

Analysis of phage genomes revealed the existence of 49 ORFs, 75 promoter sequences, and 7 rho-independent terminator sequences in P2 phage; whereas, vB_AhydM-H1 had 72 ORFs, 108 promoters, and 15 terminators (Table 1). Similar to vB_AhydM-H1, where 36% (26 ORFs) were functionally characterized with one tRNA gene (Supplementary Table S5), 33% (16 ORFs) of P2 ORFs were predicted to have functions, lacking tRNA genes (Supplementary Table S4). The remaining ORFs in both phages encoded proteins with hypothetical functions. Analysis of functional genes in both phages revealed similar distributions: phage DNA replication and repair (7 ORFs in P2, 7 ORFs in vB_AhydM-H1) and structural/packaging proteins (5 ORFs in P2, 8 ORFs in vB_AhydM-H1) constituted the major functional categories. Additionally, genes involved in host lysis, phage DNA transcription, and other functions were also present. Notably, vB_AhydM-H1 possessed an extra tRNA gene (tRNA-Gly). Both phages primarily utilized ATG (methionine) as the start codon for ORFs, with a few exceptions in P2 (ORFs 7 and 49: TTG; ORFs 12 and 27: GTG) and vB_AhydM-H1 (ORFs 26, 63, 70, and 71: GUG; ORF 15: UUG) (Supplementary Tables S4 and S5). Genes crucial for phage functions, such as DNA replication, repair, and structural components, were clustered together in the genome, often interspersed with ORFs of unknown function (Fig. 5A and B).

FIG. 5.

Proksee-derived schematic map of circular genome of A. hydrophila phages (A) P2 and (B) vB_AhydM-H1. The innermost rings show genome location, GC skew + (green) and-(purple). Two the most external rings show identified open reading frames (dark blue arrows) and results of genome annotation process.

Analysis of both phages using DeepTMHMM revealed that three hypothetical proteins (ORFs: 6, 11, and 23) of the P2 phage were predicted to have one transmembrane domain (TMD), while one hypothetical protein (ORF 17) was predicted to have two TMDs (Supplementary Fig. S2); whereas, four proteins (ORFs: 12, 43, 59, and 68) within the vB_AhydM-H1 phage were anticipated to possess a single transmembrane domain (Supplementary Fig. S2). No genes encoding virulence factors, toxins, antibiotic resistance, transposable elements, or temperate life cycle components (such as integrases, repressors, transposases, and excisionases) were identified in either of these genomes (Table 1).

Phylogenetic analysis

BLASTn was used to determine the sequence identity of the two phage genomes to other phage genomes in the NCBI nucleotide (nr/nt) database (Table 1). VIRIDIC analysis identified significant intergenomic similarities between phages: P2 exhibited 91.3%, 91.4%, and 93.3% similarity with A. hydrophila phages CF7, Ahp1, and AHPMCC7, respectively, while vB_AhydM-H1 shared 70.9%, 58.4%, and 46.6% similarity with Aeromonas phages BUCT696, pAh6-C, and PVN04, respectively (Supplementary Fig. S3). VICTOR analysis revealed that A. hydrophila phages (P2, CF7, Ahp1, AHPMCC7) formed a distinct distant cluster as compared with other Aeromonas phages (vB_AhydM-H1, BUCT696, pAh6-C). Notably, P2, Ahp1, and AHPMCC7 formed the same species, while CF7 belonged to a separate species within the same genus Ahphunavirus (Supplementary Fig. S4). Conversely, vB_AhydM-H1, BUCT696, pAh6-C, and PVN04 were classified as distinct species within the same genus Pahsextavirus (Supplementary Fig. S4). Thus, phage vB_AhydM-H1 is a novel phage related to members of the genus Pahsextavirus of the family Chaseviridae. The OPTSIL program estimated taxon boundaries at the species, genus, and family levels, using an F-value (fraction of links required for cluster fusion) of 0.5, indicating the presence of 33 species, 13 genera, and 7 family-level clusters in the phylogenetic tree (Supplementary Fig. S4).

For evaluating the phylogenetic relationship between P2 and vB_AhydM-H1 with known phages, the ViPTree server version 1.9 was employed to construct a proteomic tree derived from genome-wide sequence similarities computed through tBLASTx (Fig. 6A). P2 aligned with members of the family Autographiviridae infecting the phylum Pseudomonadota, while vB_AhydM-H1 aligned with members of the family Chaseviridae also infecting the phylum Pseudomonadota. The linear whole genome comparison diagram, demonstrating tBLASTx pairwise similarities between the most closely related genomes, indicated that P2 formed a separate cluster with Aeromonas phages CF7 and Ahp1 (Fig. 6B). Both these phages infect A. hydrophila strains and belong to the family Autographiviridae. Phage vB_AhydM-H1 clustered with Aeromonas phages BUCT696 and pAh6-C, which belong to the family Chaseviridae (Fig. 6C). The phylogenetic relationship of phages P2 and vB_AhydM-H1 with their nearest neighbors, based on tBLASTx-derived proteomic tree, is congruent with whole-genome-based phylogenetic trees (Fig. 7).

FIG. 6.

Proteomic tree generated by ViPTree of A. hydrophila phages P2 and vB_AhydM-H1. (A) Combined circular proteomic tree of phages P2 and vB_AhydM-H1, top BLASTn hits, and related phages of RefSeq genomes. Rectangular trees representing a subset of the closely related phages to P2 (B) and vB_AhydM-H1 (C) from circular tree.

FIG. 7.

Whole-genome phylogenetic tree of A. hydrophila phages P2 and vB_AhydM-H1 (highlighted with the red color outline) with other Aeromonas phages in the NCBI database. Bootstrap values have been used for branch coloring. Bootstrap value for each node has also been shown. Phages have been classified at family (F), genus (G) and species (S) levels based on the nucleotide sequence similarities. The scale bar shows 0.09 substitutions per nucleotide. Supplementary Table S3 can be referred for more details about phage name, genome size, genome type, accession no. and country of isolation.

Discussion

The emergence of antibiotic resistance in A. hydrophila strains linked to both aquaculture^39–40 and human infections⁴¹ necessitates the urgent development of alternative treatment strategies capable of replacing existing antibiotics. P2 and vB_AhydM-H1 phages, isolated from sewage, displayed high host specificity toward their isolation hosts viz. A. hydrophila, exhibiting no activity against other Aeromonas or non-Aeromonas strains. This narrow range, a common trait in Aeromonas phages,⁴² limits their use alone but allows for their potential in combination therapies (cocktails) and ensures minimal disruption to the normal flora of the host or food during therapy.⁴³ Moreover, they can be used for customized phage cocktails, phage typing, and delivery vehicles for targeted therapies. Further research is required to deliberately expand their host range by phage training or phage engineering.

TEM analysis demonstrated that the morphology of the P2 phage classified it as a podovirus, characterized by its icosahedral head and short tail. Conversely, vB_AhydM-H1 exhibited symmetrical icosahedral heads and straight, contractile tails, indicative of a myovirus. The proteomic tree derived from tBLASTx (ViPTree) revealed that the P2 phage clusters with Aeromonas phages Ahp1 and CF7, belonging to the genus Ahphunavirus within the family Autographiviridae. Conversely, phage vB_AhydM-H1 aligns with A. hydrophila phages BUCT696, pAh6-C, and pAh6.2TG, which are members of the family Chaseviridae. These findings were corroborated by analyzing both genomes using the AI-driven online software platform PhageAI,³⁷ confirming that P2 phage is classified under the Ahphunavirus genus within the Autographiviridae family, while vB_AhydM-H1 is affiliated with the Chaseviridae family. This classification is consistent with the grouping of other Aeromonas phages according to genomic relationships.^44–45 A. hydrophila phage Ahp1, earlier classified as a member of phiKMV-like subgroup of Podoviridae family,⁴⁶ was reclassified into the newly designated Ahphunavirus genus due to recent advancements in genome-based bacteriophage classification.⁴⁷ Subsequently, A. hydrophila phages, CF7, AHPMCC7, and LAh1, have also been classified as members of genus Ahphunavirus.⁴⁴ Recently, A. hydrophila phages, pAh6.2TG (MZ336020),⁴⁵ BUCT696 (OL770365),⁴⁸ pAh6-C (KJ858521),⁴⁹ and PVN04 (MW380984),⁵⁰ have also been assigned to the Chaseviridae family, exhibiting myovirus morphology, a 52–56 kb double-stranded DNA genome, and a G + C content ranging from 39.3% to 52.5%.⁵¹ Phylogenetic analyses, based on whole genomes (VICTOR analysis) and tBLASTx-derived proteomic tree, also demonstrated the clustering of vB_AhydM-H1 and pAh6.2TG phages within the same clade.

Beyond various structural, packaging proteins, and genes involved in DNA replication and repair, both phages encode putative lysins responsible for host cell lysis and the subsequent release of progeny viral particles. Lytic transglycosylases (P2:ORF4 and H1:ORF46) specifically cleaves the β-1,4 glycosidic bond in the bacterial peptidoglycan, ultimately causing cell wall degradation.⁵² Endolysin (P2:ORF10) are phage-produced enzymes that function within the bacterial host. At the end of the phage replication cycle, these enzymes degrade the host’s peptidoglycan from within, leading to cell lysis and the release of progeny virions. Because of their ability to dismantle the bacterial cell wall and kill the bacteria, lysins have recently gained significant interest as potential novel antimicrobial agents. This offers a promising approach to combat the emerging challenge of antimicrobial resistance in bacteria.

Interestingly, the ORF encoding ribonucleotide reductase was only present in the H1 phage, not in P2. Previous studies have reported a higher prevalence of ribonucleotide reductase in Myoviridae phages, while its presence is negligible in Podoviridae phages.⁵³ Furthermore, the HD domain-containing protein (ORF10) identified in H1 is also present in Aeromonas phage pAh6-C, another member of the Pahsextavirus genus.

Both phages share the genes (P2:ORF5 and H1:ORF33) for tail spikes, the structures crucial for the host recognition and attachment. H1 also encodes a portal protein (ORF19) forming a head–tail junction for DNA passage during assembly and release. Tail proteins (P2:ORF49, P2:ORF5, H1:ORF38, 39, and 41) aid in phage adhesion and injection of phage DNA in host cell. A high degree of homology was observed between the tail proteins of the studied phages and those of other Aeromonas phages belonging to their respective genera. While no specific function could be attributed to several hypothetical proteins, they exhibited notable resemblances to hypothetical proteins found in other phages. No significant sequence similarity was observed between P2 and vB_AhydM-H1 during BLASTn and VIRIDIC analyses (0.1% intergenomic similarity), as well as VICTOR analysis.

The latent period and burst size of phages are critical determinants in biocontrol selection. Studies have shown that a shorter latent period and higher burst size enhance bacterial inactivation.⁵⁴ Phage P2 showed an adsorption rate of ∼90%, 90-minute latent period, and 34 ± 7 PFU/cell burst size, while vB_AhydM-H1 has similar adsorption but a 120-min latent period and a larger burst size of 179 ± 12 PFU/host cell. Aeromonas phages exhibit diverse latent periods (15–150 min) and burst sizes (2–626 PFU/host cell).⁵⁵ Correlations between latent period and burst size remain unclear.^56,57 Factors like host growth rate and medium composition influence burst size and latent period of phages.⁵⁸ A longer latent period can result in larger burst sizes by allowing more time for progeny maturation.⁵⁹

Understanding phage stability under various environmental stresses is crucial for their use in laboratory or field settings, and long-term storage. Research shows that phages remain active across a pH range from 4 to 12 after 3 h of incubation. Although aquaculture and food environments usually have moderate temperatures and pH levels that do not drastically affect phage activity, production and formulation processes may involve harsher conditions. Therefore, selecting phages with stability across different temperatures and pH levels is recommended for optimal production.

Conclusions

This study identified and characterized two novel A. hydrophila phages isolated from sewage. Phage P2 belongs to the Ahphunavirus genus, while vB_AhydM-H1 belongs to the Pahsextavirus genus. Interestingly, both tBLASTx-derived proteomic and whole-genome phylogenetic analyses demonstrated highly consistent topological arrangements. Both phages exhibited consistent characteristics in host range, morphology, adsorption rate, latent period, and burst size. They infect only one A. hydrophila strain, have high latent periods, and tolerate a broad range of pH and temperature. Importantly, they lack genes associated with toxins, antibiotic resistance, or temperate life cycles, making them promising for biocontrol in food. Further research on their holin and endolysin genes could yield potent antimicrobial proteins, while engineering bacteriophages can improve their host range and lysis capabilities, presenting a promising avenue for developing targeted and effective therapies against Aeromonas infections.

Consent for Publication

All listed authors have approved the article before submission, including the names and order of authors.

Authors’ Contributions

V.N. conceptualized, designed, and performed the experiments; analyzed data; and wrote and reviewed the article. L.P.G. contributed in experiments and data analysis. S.N. reviewed data and article. A.T. was responsible for analyzing data and editing the article. All authors read and approved the final article.

Author Disclosure Statement

The authors declare no conflicts of interest.

Funding Information

This work was funded by the Department of Atomic Energy, India.