Isolation Of Bacteriophages Research Articles

Improving high-throughput techniques for bacteriophage discovery in multi-well plates

Bacteriophages (also called phages) are viruses of bacteria that have numerous applications in medicine, agriculture, ecology, and molecular biology. With the increasing interest in phages for their many uses, it is now especially important to make phage discovery more efficient and economical.Using the host Mycobacterium smegmatis mc2155, which is a model organism for phage discovery research and is closely related to important pathogens of humans and other animals, we investigated three procedures that are an integral part of phage discovery: enrichment of environmental samples, phage isolation and detection (which can also be used for host range determination), and phage purification. Enrichment in 6-well plates was successful with most environmental samples, and enrichment in 24- and 96-well plates was successful with some environmental samples, demonstrating that larger sample volumes are preferred when possible, but smaller sample volumes may be acceptable if the starting concentration of phages is sufficiently high. Measuring absorbance in multi-well plates was at least as sensitive as the traditional plaque assay for the detection of phages. We also demonstrated a technique for the purification of single phage types from mixed cultures in liquid medium.Multi-well techniques can be used as alternatives or complementary approaches to traditional methods of phage discovery and characterization depending on the needs of the researcher in terms of time, available resources, host species, phage-bacteria matches, and specific goals. In the future, these techniques could be applied to the discovery of phages of aquatic mycobacteria and other hosts for which few phages have currently been isolated.

Morphological and Genetic Characterization of Eggerthella lenta Bacteriophage PMBT5.

Eggerthella lenta is a common member of the human gut microbiome. We here describe the isolation and characterization of a putative virulent bacteriophage having E. lenta as host. The double-layer agar method for isolating phages was adapted to anaerobic conditions for isolating bacteriophage PMBT5 from sewage on a strictly anaerobic E. lenta strain of intestinal origin. For this, anaerobically grown E. lenta cells were concentrated by centrifugation and used for a 24 h phage enrichment step. Subsequently, this suspension was added to anaerobically prepared top (soft) agar in Hungate tubes and further used in the double-layer agar method. Based on morphological characteristics observed by transmission electron microscopy, phage PMBT5 could be assigned to the Siphoviridae phage family. It showed an isometric head with a flexible, noncontractile tail and a distinct single 45 nm tail fiber under the baseplate. Genome sequencing and assembly resulted in one contig of 30,930 bp and a mol% GC content of 51.3, consisting of 44 predicted protein-encoding genes. Phage-related proteins could be largely identified based on their amino acid sequence, and a comparison with metagenomes in the human virome database showed that the phage genome exhibits similarity to two distantly related phages.

A Perspective on Studies of Phage DNA Packaging Dynamics

The Special Issue “DNA Packaging Dynamics of Bacteriophages” is focused on an event that is among the physically simplest known events with biological character. Thus, phage DNA (and RNA) packaging is used as a relatively accessible model for physical analysis of all biological events. A similar perspective motivated early phage-directed work, which was a major contributor to early molecular biology. However, analysis of DNA packaging encounters the limitation that phages vary in difficulty of observing various aspects of their packaging. If a difficult-to-access aspect arises while using a well-studied phage, a counterstrategy is to (1) look for and use phages that provide a better access “window” and (2) integrate multi-phage-accessed information with the help of chemistry and physics. The assumption is that all phages are characterized by the same evolution-derived themes, although with variations. Universal principles will emerge from the themes. A spin-off of using this strategy is the isolation and characterization of the diverse phages needed for biomedicine. Below, I give examples in the areas of infectious disease, cancer, and neurodegenerative disease.

Two Newly Isolated Enterobacter-Specific Bacteriophages: Biological Properties and Stability Studies.

In an era of antibiotic therapy crisis caused by spreading antimicrobial resistance, and when recurrent urinary tract infections constitute a serious social and medical problem, the isolation and complex characterization of phages with a potential therapeutic application represents a promising solution. It is an inevitable, and even a necessary direction in the development of current phage research. In this paper, we present two newly isolated myoviruses that show lytic activity against multidrug-resistant clinical isolates of Enterobacter spp. (E. cloacae, E. hormaechei, and E. kobei), the genomes of which belong to a poorly represented phage group. Both phages were classified as part of the Tevenvirinae subfamily (Entb_43 was recognized as Karamvirus and Entb_45 as Kanagawavirus). Phage lytic spectra ranging from 40 to 60% were obtained. The most effective phage-to-bacteria ratios (MOI = 0.01 and MOI = 0.001) for both the phage amplification and their lytic activity against planktonic bacteria were also estimated. Complete adsorption to host cells were obtained after about 20 min for Entb_43 and 10 min for Entb_45. The phage lysates retained their initial titers even during six months of storage at both −70 °C and 4 °C, whereas storage at 37 °C caused a complete loss in their activity. We showed that phages retained their activity after incubation with solutions of silver and copper nanoparticles, which may indicate possible synergistic antibacterial activity. Moreover, a significant reduction in phage titers was observed after incubation with a disinfectant containing octenidinum dihydrochloridum and phenoxyethanol, as well as with 70% ethanol. The observed maintenance of phage activity during incubation in a urine sample, along with other described properties, may suggest a therapeutic potential of phages at the infection site after intravesical administration.

ISOLATION AND CHARACTERISTICS OF BACTERIOPHAGES SANITARY-INDICATIVE MICROORGANISMS

Isolated phages showed activity toward opportunistic and pathogenic bacteria of genera Escherichia (K3P1, K3P2, BIM BV-44 D) and Pseudomonas (PsP1), formed lysis zones shaped as negative homogeneous colonies with a clear-cut even edge (K3P1, PsP1, BIM BV -44 D), or negative colonies with a peripheral zone of partial lysis (K3P2). The diameter of phage plaques varied from 0,5–1,5 mm. Lytic activity spectrum of phage isolates was investigated. It was found that the studied bacteriophages displayed species specificity. Bacteriophages K3P1, K3P2 were distinguished by the highest lytic range. Differences in the isolated bacteriophages according to restriction profiles were revealed. Bacteriophage distinctions in restriction profiles were revealed. Interaction of bacteriophages with cells of bacteria of E. coli and Ps. aeruginosa was explored. The tested bacteriophage strains demonstrating lytic activity toward sanitary-indicative bacterial species may find use as disinfectants in detergents.

Isolation and Molecular Characterization of a Novel Lytic Bacteriophage That Inactivates MDR Klebsiella pneumoniae Strains.

The worldwide increase in serious infections caused by multidrug-resistant (MDR) K. pneumoniae emphasizes the urgent need of new therapeutic strategies for the control of this pathogen. There is growing interest in the use of bacteriophages (or phages) to treat K. pneumoniae infections, and newly isolated phages are needed. Here, we report the isolation and physical/biological/molecular characterization of a novel lytic phage and its efficacy in the control of MDR K. pneumoniae. The phage vB_KpnS_Uniso31, referred to hereafter as phage Kpn31, was isolated from hospital wastewater using K. pneumoniae CCCD-K001 as the host. Phage Kpn31 presents a siphovirus-like morphotype and was classified as Demerecviridae; Sugarlandvirus based on its complete genome sequence. The 113,444 bp Kpn31 genome does not encode known toxins or antimicrobial resistance genes, nor does it encode depolymerases related sequences. Phage Kpn31 showed an eclipse time of 15 min and a burst size of 9.12 PFU/host cell, allowing us to conclude it replicates well in K. pneumoniae CCCD-K001 with a latency period of 30 min. Phage Kpn31 was shown to be effective against at least six MDR K. pneumoniae clinical isolates in in vitro antibacterial activity assays. Based on its features, phage Kpn31 has potential for controlling infections caused by MDR K. pneumoniae.

Characterization and Genomic Analysis of ssDNA Vibriophage vB_VpaM_PG19 within Microviridae, Representing a Novel Viral Genus.

ABSTRACTVibrio parahaemolyticus, a widespread marine bacterium, is responsible for a variety of diseases in marine organisms. Consumption of raw or undercooked seafood contaminated with V. parahaemolyticus is also known to cause acute gastroenteritis in humans. While numerous dsDNA vibriophages have been isolated so far, there have been few studies of vibriophages belonging to the ssDNA Microviridae family. In this study, a novel ssDNA phage, vB_VpaM_PG19 infecting V. parahaemolyticus, with a 5,572 bp ssDNA genome with a G+C content of 41.31% and encoded eight open reading frames, was isolated. Genome-wide phylogenetic analysis of the total phage isolates in the GenBank database revealed that vB_VpaM_PG19 was only related to the recently deposited vibriophage vB_VpP_WS1. The genome-wide average nucleotide homology of the two phages was 89.67%. The phylogenetic tree and network analysis showed that vB_VpaM_PG19 was different from other members of the Microviridae family and might represent a novel viral genus, together with vibriophage vB_VpP_WS1, named Vimicrovirus. One-step growth curves showed that vB_VpaM_PG19 has a short incubation period, suggesting its potential as an antimicrobial agent for pathogenic V. parahaemolyticus.IMPORTANCE Vibriophage vB_VpaM_PG19 was distant from other isolated microviruses in the phylogenetic tree and network analysis and represents a novel microviral genus, named Vimicrovirus. Our report describes the genomic and phylogenetic features of vB_VpaM_PG19 and provides a potential antimicrobial candidate for pathogenic V. parahaemolyticus.

Formulation of a Novel Phage Cocktail against Vibrio cholerae O1

A number of 26 phages lytic for 26 isolates of V. cholerae O1, biotype El Tor, Serotype Inaba are isolated from sewage water. The phage isolates showed host range of 35-65% against V. cholerae O1 by spot lysis methods. The morphological properties of plaques formed on the top agarose are studied and showed that most virulent phages had larger diameter (in millimeter) with regular or irregular margin cut and clear plaque comparing with smaller diameter and turbid plaque for those less virulent phages. A total of 8 phages are selected for formulation the phage cocktail in order to extend the host-range of phages in cocktail collectively. The %inhibition assay for every single phage of the 8 phages selected for formulation of phage cocktail ranged from 0-75, whereas that of the 8 phages cocktail was 100%inhibition against all V. cholerae O1 isolates. The formulation of the 8 phages in a cocktail proved to an effective approach to achieve the broad host-range activity towards V. cholerae O1 isolates and made it possible to go forwards the animal model for further studies on phage therapy for animal model of cholera.

Phage-host coevolution in natural populations.

Coevolution between bacteriophages (phages) and their bacterial hosts occurs through changes in resistance and counter-resistance mechanisms. To assess phage-host evolution in wild populations, we isolated 195 Vibrio crassostreae strains and 243 vibriophages during a 5-month time series from an oyster farm and combined these isolates with existing V. crassostreae and phage isolates. Cross-infection studies of 81,926 host-phage pairs delineated a modular network where phages are best at infecting co-occurring hosts, indicating local adaptation. Successful propagation of phage is restricted by the ability to adsorb to closely related bacteria and further constrained by strain-specific defence systems. These defences are highly diverse and predominantly located on mobile genetic elements, and multiple defences are active within a single genome. We further show that epigenetic and genomic modifications enable phage to adapt to bacterial defences and alter host range. Our findings reveal that the evolution of bacterial defences and phage counter-defences is underpinned by frequent genetic exchanges with, and between, mobile genetic elements.

Initial Survey of Staphylococcus Bacteriophages and Simple Modifications in the Enrichment Method for Enhancing Plaques

Isolation of bacteriophages against Staphylococcus aureus is difficult when compared to the bacteriophages of other genera. We did a preliminary survey for the presence of bacteriophages of S. aureus and Escherichia coli in the environmental water samples. We did not get bacteriophages against S. aureus, but bacteriophages against E. coli were ample. Subsequently, a river-water sample was taken and through the enrichment method with some modifications, we got a bacteriophage against a methicillin-resistant S. aureus. Here, we are reporting simple changes in the enrichment method to enhance plaques that have an extremely tiny and turbid morphology. This can be useful for researchers facing difficulty in finding lytic bacteriophages from water samples.

Characterization and genomic analysis of the novel Aeromonas veronii phage pAEv1812.

Bacteria of the species Aeromonas veronii are generally ubiquitous in different aquatic environments and are capable of causing a variety of diseases in aquatic animals. In this work, the phage isolate pAEv1812, which is lytic to A. veronii strain 1810, was characterized and sequenced. Morphological analysis revealed that pAEv1812 has a symmetrical head and a long non-contractile tail. A one-step growth curve analysis showed that the latent period and burst period of pAEv1812 were approximately 40 min and 80 min, respectively, with a burst size of 190 PFU/infected cell. The genome of phage pAEv1812 is 61,461 bp in length with a G+C content of 61.4%. Eighteen of the 75 putative proteins encoded by this phage have known functions, and there were no rRNA and tRNA genes in the genome. Phylogenetic analysis based on amino acid sequences of the major capsid protein and terminase large subunit suggested that phage pAEv1812 should be included as a member of the genus Chivirus.

Isolation of a lytic bacteriophage against extensively drug-resistant Acinetobacter baumannii infections and its dramatic effect in rat model of burn infection.

Objectives Acinetobacter Baumannii is an opportunistic nosocomial pathogen belonging to the Moraxellaceae family. The emergence of multidrug resistant strains of this pathogen caused many problems for hospitals and patients. The aim of the current study was to isolate, identify, and morphologically, physiologically, and in vivo analyze a new lytic bacteriophage targeting extensively drug‐resistant (XDR) A. baumannii.Materials and MethodsDifferent wastewater samples were tested for isolation of lytic bacteriophage against 19 A. baumannii isolates obtained from patients hospitalized in a hospital in Arak, Iran, from January 2019 to March 2019. The phenotypic and genotypic characteristics of A. baumannii strains (resistance genes including: adeA, adeB, adeC, adeR, adeS, ISAba1, blaOXA‐23, blaOXA‐24) were analyzed. The isolated phage characteristics including adsorption time, pH and thermal stability, host range, one‐step growth rate, electron microscopy examination, and therapeutic efficacy of the phage were also investigated. Therapeutic efficacy of the phage was evaluated in a rat model with burn infection of XDR A. baumannii. The lesion image was taken on different days after burning and infection induction and was compared with phage untreated lesions.ResultsThe results showed unique characteristics of the isolated phage (vB‐AbauM‐Arak1) including high specificity for Acinetobacter baumannii, stability at a relatively wide range of temperatures and pH values, short adsorption time, short latent period, and large burst size. In relation to the therapeutic efficacy of the phage, the lesion area decreased in phage‐treated groups over 14 days than in those untreated, significantly (p < 0.05).ConclusionOur findings demonstrated that isolated lytic phage was able to eliminate burn infections caused by XDR A. baumannii in a rat model. So, it may be recommended as alternative options toward to developing a treatment for extensively drug resistant Acinetobacter infections.

Investigation of the Phageome and Prophages in French Cider, a Fermented Beverage.

Phageomes are known to play a key role in the functioning of their associated microbial communities. The phageomes of fermented foods have not been studied thoroughly in fermented foods yet, and even less in fermented beverages. Two approaches were employed to investigate the presence of phages in cider, a fermented beverage made from apple, during a fermentation process of two cider tanks, one from an industrial producer and one from a hand-crafted producer. The phageome (free lytic phages) was explored in cider samples with several methodological developments for total phage DNA extraction, along with single phage isolation. Concentration methods, such as tangential flow filtration, flocculation and classical phage concentration methods, were employed and tested to extract free phage particles from cider. This part of the work revealed a very low occurrence of free lytic phage particles in cider. In parallel, a prophage investigation during the fermentation process was also performed using a metagenomic approach on the total bacterial genomic DNA. Prophages in bacterial metagenomes in the two cider tanks seemed also to occur in low abundance, as a total of 1174 putative prophages were identified in the two tanks overtime, and only two complete prophages were revealed. Prophage occurrence was greater at the industrial producer than at the hand-crafted producer, and different dynamics of prophage trends were also observed during fermentation. This is the first report dealing with the investigation of the phageome and of prophages throughout a fermentation process of a fermented beverage.

Isolation and Characterization of Lytic Bacteriophages from Wastewater with Phage Therapy Potentials Against Gram-Negative Bacteria.

The increase of multidrug resistance in bacteria has increased the efforts in search of alternative methods. The aim of the present study was to isolate and characterize the lytic phages and assess their lytic activity against a number of gram-negative bacteria. The phages and their respective hosts were isolated from wastewater collected from the municipal sewer system of Trabzon, Turkey. The lytic activities of phage were determined using the agar spot test. The identification and antibiotic susceptibility of host bacteria were determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Phoenix 100, respectively. The phages were characterized morphologically using transmission electron microscopy. One of the phages, Enteroc21, which has a broad-host-range, was further characterized by genome restriction endonuclease analysis and burst size. Two phages infected strains of four different species, nine phages were able to infect 2-4 strains belonging to one or two species, and three phages showed lytic activity against only the hosts from which they were isolated. All phages belonged to the Siphoviridae, Myoviridae, and Podoviridae family based on trans- mission electron microscopy morphology. The Enteroc21 had more than 100 kb genome size and a burst size of 180 per infected cell. Most of the host strains were resistant to ampicillin, amoxicillin-clavulanic acid, and in particular, Achromobacter xylosoxidans TRAX 13 was multidrug-resistant showing resistance to cefepime, aztreonam, gentamicin, netilmicin, and ciprofloxacin. This study showed that the isolated phages have the potential to be used in phage therapy against various bacterial infections, including multidrug-resistant bacteria.

Isolation of Nine Bacteriophages Shown Effective against Erwinia amylovora in Korea.

Erwinia amylovora is a devastating bacterial plant pathogen that infects Rosaceae including apple and pear and causes fire blight. Bacteriophages have been considered as a biological control agent for preventing bacterial infections of plants. In this study, nine bacteriophages (ΦFifi011, ΦFifi044, ΦFifi051, ΦFifi067, ΦFifi106, ΦFifi287, ΦFifi318, ΦFifi450, and ΦFifi451) were isolated from soil and water samples in seven orchards with fire blight in Korea. The genetic diversity of bacteriophage isolates was confirmed through restriction fragment length polymorphism pattern analysis. Host range of the nine phages was tested against 45 E. amylovora strains and 14 E. pyrifoliae strains and nine other bacterial strains. Among the nine phages, ΦFifi044 and ΦFifi451 infected and lysed E. amylovora only. And the remaining seven phages infected both E. amylovora and E. pyrifoliae. The results suggest that the isolated phages were different from each other and effective to control E. amylovora, providing a basis to develop biological agents and utilizing phage cocktails.

Isolation and Characterization of Lytic Bacteriophage against Methicillin Resistant Staphylococcus aureus from Pyoderma in Dog

Background: Pyoderma in dog poses complexity owing to involvement of multi drug resistant bacterial infection particularly Staphylococcus species. This may result into failure of antibiotic therapy in pyoderma. Therapeutic use of lytic bacteriophage (phage) may be an alternative to deal antimicrobial resistance in veterinary medicine. There is no report available on isolation and characterization of bacteriophage from clinical cases of pyoderma in animals, hence the study was performed with aim to isolate and characterize lytic phage against methicillin resistant S. aureus (MRSA). Methods: The phage was isolated from pus sample of dog suffering from severe pyoderma. The phage was isolated using MRSA as a bacterial host and was named as Staphylococcus phage BVC1 (SPBVC1). The morphology of the phage was determined using TME and was characterised by determining the host range and lytic potential of the phage at range of temperature and pH. Result: The morphology of phage revealed an icosahedral head of diameter 81.31 nm. with sheath and a central tube and a tail of 92.08 nm. It showed strong lytic activity against Methicillin resistant S. aureus and was stable under a range of temperature varying from 4oC to 45oC and pH from 4 to 11. The phage has shown lytic activity against the MRSA however no lytic activity against the MSSA was shown by the phage. The high specificity of the phage for MRSA indicated its potential use as an alternative therapeutic approach against multidrug resistant staphylococcal infections.

Poly(epsilon-lysine) dendrons and nucleic acids complexes for non-viral delivery of bacteriophage DNA into bacterial cells

Phage therapy has been advocated as an alternative to antibiotics in the treatment of bacterial infections. However, this approach can be affected by batch-to-batch variability in the harvesting, isolation and storage of specie-specific bacteriophages. The present paper presents the development of synthetic nanostructured carriers that can form complexes with isolated bacteriophage DNA, while ensuring the penetration of the carrier through bacterial wall and membrane. The complexation capability of these poly(epsilon-lysine) dendrons with bacteriophage DNA was tested by mixing the hyperbranched molecules at various charge ratios (132:1 to 0.06:1 of positively charged carriers to negatively charged DNA). These electrostatic complexes were assessed by gel retardation, ethidium bromide displacement assay and transmission electron microscopy. The self-assembly of poly(epsilon-lysine) dendrons with the nucleic acids led to charge ratio-dependent electrostatic complexes. Transfection of both complexed and naked DNA in Proteus mirabilis bacteria demonstrated that the viral DNA maintained its replicative ability with the formation of whole viral particles. Additionally, the dendron’s cytotoxicity was assayed by lactate dehydrogenase (LDH) release and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) reduction assays. In both assays, 15% cytotoxicity and even lower (6%) necrotic effects were observed by MTT and LDH respectively. Overall, these findings demonstrate that it is possible to complex bacteriophage DNA with poly(epsilon-lysine) dendrons, of three branching generations (Gen3K), and suggests that Gen3K has the potential to be used as a reliable alternative to antibiotics.

Molecular dissection of the first Staphylococcus cohnii temperate phage IME1354_01

In staphylococcal phage research, studies specific to coagulase-negative staphylococci (CoNS) remain severely under-represented, and the number of temperate bacteriophages is limited. This investigation identifies a novel temperate phage IME1354_01 from the strain Staphylococcus cohnii IME1354, which was isolated from the skin of a patient with foot ulcer disease. The phage IME1354_01 is the first isolated temperate phage of S. cohnii, and was determined to have a long-tail morphology using TEM. Its genome was found to be a 42,706-bp linear dsDNA molecule with a GC content of 34%. The integration of IME1354_01 occurred using a tRNA-Ser coding gene, and it did not affect tRNA-Ser function. The genome of IME1354_01 is most closely related to that of the temperate Staphylococcus arlettae phage vB_Sars_BM31 with 10% homology coverage and 83.73% nucleotide identity. In addition, they showed similarities mainly in the DNA replication, DNA packaging and partial morphogenesis modules. We propose that a new genus should be created for IME1354_01 based on the intergenomic similarities (maximum is 23%) obtained from the VIRIDIC calculations. The isolation and in-depth study of the novel phage, IME1354_01, will improve our understanding of the evolutionary relationship between temperate phages and their hosts.

Diversity of lytic bacteriophages against XDR Klebsiella pneumoniae sequence type 16 recovered from sewage samples in different parts of the world

Bacteriophages (phages) are viruses considered to be natural bacterial predators and widely detected in aquatic environments. Sewage samples are an important source of phage isolation since high density and diversity of bacterial cells are present, due to human, animal and household fluids. This study aims to investigate and characterise phages against an extremely drug-resistant (XDR) lineage, Klebsiella pneumoniae ST16, using sewage samples from different parts of the World. Sewage samples from Brazil, Bangladesh, Saudi Arabia, Thailand and the United Kingdom were collected and used to investigate phages against ten K. pneumoniae ST16 (hosts) recovered from infection sites. The phages were microbiological and genetically characterised by double-agar overlay (DLA), transmission electron microscopy and Illumina WGS. The host range against K. pneumoniae belonging to different sequence types was evaluated at different temperatures by spot test. Further phage characterisation, such as efficiency of plating, optimal phage temperature, and pH/temperature susceptibility, were conducted. Fourteen lytic phages were isolated, belonging to Autographiviridae, Ackermannviridae, Demerecviridae, Drexlerviridae, and Myoviridae families, from Brazil, Bangladesh, Saudi Arabia and Thailand and demonstrated a great genetic diversity. The viruses had good activity against our collection of clinical K. pneumoniae ST16 at room temperature and 37 °C, but also against other important Klebsiella clones such as ST11, ST15, and ST258. Temperature assays showed lytic activity in different temperatures, except for PWKp18 which only had activity at room temperature. Phages were stable between pH 5 and 10 with minor changes in phage activity, and 70 °C was the temperature able to kill all phages in this study. Using sewage from different parts of the World allowed us to have a set of highly efficient phages against an K. pneumoniae ST16 that can be used in the future to develop new tools to combat infections in humans or animals caused by this pathogen.

Isolation and characterization of Aeromonas hydrophila-specific lytic bacteriophages

Aeromonas hydrophila can infect a broad range of fish living in freshwater and estuaries. Because the majority of A. hydrophila is resistant to antimicrobials, alternative treatment strategies should be developed. Bacteriophage may be an alternate approach to control infectious diseases. For this reason, we isolated AP-T65, AP-T5, AP-Y28, and AP-AT phages and assessed their lytic activity against A. hydrophila strains, described them phenotypically, kinetically, and genetically, and evaluated their cytotoxicity in vitro and in vivo assays. After sequencing the major capsid protein (MCP) gene of the phages, it was found that all phages belonged to the Myroviridae family. Phages demonstrated lytic activity against Aeromonas sp., A. salmonicida, A. veronii, and A. media. Phages had a short latent period of roughly 10–15 min and the burst sizes of all Aeromonas phages were found to be between 10 and 20 plaque forming units (PFU)/infected cell. The AP-T65, AP-T5, AP-Y28, and AP-AT phages survived a wide range of pH values ranging from 3 to 10, as well as temperatures ranging from 4 to 37 °C. Aeromonas phages showed no impact on fish or EPC cell lines. Therefore, Aeromonas phages are safe and they can be used as a cocktail to treat A. hydrophila infection. Because of their short latent period and greater burst size, phages isolated in the present study would be ideal candidates for phage therapy, and they might be used as a cocktail for improved control effects. The application of a cocktail of these phages may also prevent the formation of phage-resistant bacteria.