Aeromonas Phage Research Articles

Isolation, characterization and functional analysis of a bacteriophage targeting Culex pipiens pallens resistance-associated Aeromonas hydrophila

BackgroundCulex pipiens pallens is a well-known mosquito vector for several diseases. Deltamethrin, a commonly used pyrethroid insecticide, has been frequently applied to manage adult Cx. pipiens pallens. However, mosquitoes can develop resistance to these insecticides as a result of insecticide misuse and, therefore, it is crucial to identify novel methods to control insecticide resistance. The relationship between commensal bacteria and vector resistance has been recently recognized. Bacteriophages (= phages) are effective tools by which to control insect commensal bacteria, but there have as yet been no studies using phages on adult mosquitoes. In this study, we isolated an Aeromonas phage vB AhM-LH that specifically targets resistance-associated symbiotic bacteria in mosquitoes. We investigated the impact of Aeromonas phage vB AhM-LH in an abundance of Aeromonas hydrophila in the gut of Cx. pipiens pallens and its effect on the status of deltamethrin resistance.MethodsPhages were isolated on double-layer agar plates and their biological properties analyzed. Phage morphology was observed by transmission electron microscopy (TEM) after negative staining. The phage was then introduced into the mosquito intestines via oral feeding. The inhibitory effect of Aeromonas phage vB AhM-LH on Aeromonas hydrophila in mosquito intestines was assessed through quantitative real-time PCR analysis. Deltamethrin resistance of mosquitoes was assessed using WHO bottle bioassays.ResultsAn Aeromonas phage vB AhM-LH was isolated from sewage and identified as belonging to the Myoviridae family in the order Caudovirales using TEM. Based on biological characteristics analysis and in vitro antibacterial experiments, Aeromonas phage vB AhM-LH was observed to exhibit excellent stability and effective bactericidal activity. Sequencing revealed that the Aeromonas phage vB AhM-LH genome comprises 43,663 bp (51.6% CG content) with 81 predicted open reading frames. No integrase-related gene was detected in the vB AH-LH genome, which marked it as a potential biological antibacterial. Finally, we found that Aeromonas phage vB AhM-LH could significantly reduce deltamethrin resistance in Cx. pipiens pallens, in both the laboratory and field settings, by decreasing the abundance of Aeromonas hydrophila in their midgut.ConclusionsOur findings demonstrate that Aeromonas phage vB AhM-LH could effectively modulate commensal bacteria Aeromonas hydrophila in adult mosquitoes, thus representing a promising strategy to mitigate mosquito vector resistance.Graphical

Isolation and Characterization of a Novel Virulent Phage ASG01 of Aeromonas salmonicida and Its Cell Wall Hydrolase Activity.

Aeromonas salmonicida is an important pathogen that causes furunculosis in trout and salmon with high morbidity and mortality, resulting in significant economic losses in aquaculture. Overuse of antibiotics has led to the continuous emergence of drug-resistant strains. Hence, there is an urgent need to find an alternative environmentally friendly antimicrobial agent. In this study, we isolated a virulent phage of A. salmonicida, named ASG01, which belongs to the Myoviridae family and maintains lytic activity at a pH value range from 4 to 12 and in the temperature range from 30 °C to 60 °C. The whole genomic sequence of ASG01 showed 82% similarity to Aeromonas phage pAh6-C. The cell wall hydrolase (Cwh)-encoding gene from the genome of ASG01 was predicted and heterologously expressed. Notably, in the absence of additional phage genes, endogenous expression of Cwh could lyse E. coli cells and greatly inhibit the growth of tested fish pathogenic bacteria. The lytic activity of Cwh was eliminated when the predicted active site was mutated. These results indicate that Cwh of ASG01 possessed excellent lytic activity and a wide antibacterial spectrum, suggesting its potential as an effective enzybiotic.

Isolation and characterization of a novel temperate bacteriophage infecting Aeromonas hydrophila isolated from a Macrobrachium rosenbergii larvae pond

Aeromonas hydrophila is an opportunistic pathogen that frequently leads to significant mortality in various commercially cultured aquatic species. Bacteriophages offer an alternative strategy for pathogens elimination. In this study, we isolated, identified, and characterized a novel temperate A. hydrophila phage, designated as P05B. The bacteriophage P05B is a myovirus based on its morphological features, and possesses the capability to lyse A. hydrophila strains isolated from shrimp. The optimal multiplicity of infection (MOI), adsorption rate, latent period, and burst size for phage P05B were determined to be 0.001, 91.7 %, 20 min, and 483 PFU/cell, respectively. Phage P05B displayed stability across a range of temperatures (28–50 °C) and pH values (4.0–10.0). Sequence analysis unveiled that the genome of phage P05B comprises 32,302 base pairs with an average G + C content of 59.4 %. A total of 40 open reading frames (ORF) were encoded within the phage P05B genome. The comparative genomic analyses clearly implied that P05B might represent a novel species of the genus Bielevirus under Peduoviridae family. A phylogenetic tree was reconstructed, demonstrating that P05B shares a close evolutionary relationship with other Aeromonas and Aeromonas phages. In conclusion, this study increased our knowledge about a new temperate phage of A. hydrophila with strong lytic ability.

Isolation and Characterization of Lytic Bacteriophages against Aeromonas dhakensis Isolated from Water in Thailand

Phage therapy has emerged as a potential solution to the issue of multidrug-resistant bacteria. In this study, a novel bacteriophage vB_AdhS_M4 , which infects Aeromonas dhakensis, was isolated. A. dhakensis strains were isolated from water samples in Thailand and identified by biochemical and 16S rDNA sequence analysis. Four isolates, namely AM, NGP8, AK3, and SBKN4 were identified as A. dhakensis and used as hosts for phage isolation. Only 1 phage, vB_AdhS_M4, was obtained using A. dhakensis AM as host. Phage vB_AdhS_M4 morphology, host range, one-step growth curve, pH and temperature stability and genome were investigated. Transmission electron microscopy revealed that vB_AdhS_M4 has an icosahedral head of 64.8 ± 0.51 nm and a long tail of 185.4 ± 0.48 nm, suggesting that it belongs to the Siphoviridae family. Phage vB_AdhS_M4 had a latent period of 50 mins and a mean burst size of approximately 48 phage particles per infected cell. Our study on host-range determination demonstrated that this phage was specific for infecting A. dhakensis. Phage vB_AdhS_M4 was stable within a pH range of 4-7 and at temperatures ranging from 4 to 45 °C. The complete genome of Aeromonas phage vB_AdhS_M4 is 61,429 bp in length, with a G+C content of 61.7% and 77 open reading frames. In vitro A. dhakensis growth inhibition were observed with vB_AdhS_M4 at various MOIs. The greatest reduction in cell count occurred during the first 6-12 h of incubation with the phage, compared to the uninfected bacterial control, at all MOIs tested. Although the bacteria regrew after 24 h of incubation with the phage, the bacterial count remained lower than that of the control throughout the entire 48-h period. Based on these findings, it appears that vB_AdhS_M4 could be a valuable tool for biocontrol of A. dhakensis in aquaculture.

Whole Genome Sequence of a Novel Bacteriophage APT65 Infecting Aeromonas hydrophila.

Aeromonas hydrophila is a prevalent pathogenic bacterium in aquaculture that causes economic loss around the world. Antimicrobials are used to control and prevent the incidence of bacterial pathogens in aquaculture. However, they lead to the emergence of antimicrobial resistance strains and the accumulation of antibiotic residues in fish tissue. To address these issues, bacteriophages may be promising alternatives to many antibiotics in combating bacterial infections in aquaculture. The phage specific to A. hydrophila was isolated from domestic wastewater. The morphology of phages was analyzed using transmission electron microscopy. The genomic DNA of the Aeromonas phage T65 strain (APT65) phage was sequenced with a paired-end read length of 2 × 150 bp. The genome sequence was assembled and annotated. The tRNAs were predicted, and antimicrobial resistance and virulence genes were screened. A representation of the APT65 genome was constructed. The genome of APT65 is linear double-stranded DNA with 85188 base pairs having 116 open reading frames (ORFs) and a G + C content of 39.41%. The 32 ORFs were predicted to encode proteins with known phage functions. No virulence factors, antibiotic resistance genes, or temperate lifestyle genes were found. The phage is icosahedral and measures 60 nm in diameter. Based on the whole genome sequence, APT65 belongs to Lahexavirus. The taxonomic analysis of the phage with a genome length of 85,188 bp revealed that it is a new species of the genus Lahexavirus. We announce the whole genome sequence of APT65, which should be named Lahexavirus APT65, as well as the absence of antimicrobial resistance and virulence factors from its genome. Based on our results, the Lahexavirus APT65 phage may have potential as a therapeutic agent to tackle antimicrobial resistance in aquaculture.

A Novel Aeromonas popoffii Phage AerP_220 Proposed to Be a Member of a New Tolavirus Genus in the Autographiviridae Family.

Aeromonas popoffii is one of the environmental Aeromonas species. A number of factors of virulence have been described for this species and it has been reported as a causative agent of urinary tract infection. The first A. popoffii bacteriophage AerP_220 along with its host strain A. popoffii CEMTC 4062 were isolated from river water. The phage has a podovirus morphotype, shows a narrow host range and is lytic against the host strain. The AerP_220 genome comprises 45,207 bp and does not contain genes responsible for antibiotic resistance and toxin production. Fifty-nine co-directional putative ORFs were found in the AerP_220 genome. Thirty-three ORFs encoded proteins with predicted functions; the products of 26 ORFs were hypothetical proteins. AerP_220 genome analysis revealed that this phage can be considered a novel species within the Autographiviridae family. Comparative genomic and proteomic analysis revealed that AerP_220 along with the Aeromonas phage vB_AspA_Tola (OM913599) are members of a new putative Tolavirus genus in the family Autographiviridae. The Gajwadongvirus and proposed Tolavirus genera along with Pantoea phage Nufs112 and phage Reminis could form a new Tolavirinae subfamily within the Autographiviridae family.

Novel Aeromonas Phage Ahy-Yong1 and Its Protective Effects against Aeromonas hydrophila in Brocade Carp (Cyprinus aka Koi).

Aeromonas hydrophila is a zoonotic pathogen and an important fish pathogen. A new lytic phage, Ahy-yong1, against multi-antibiotic-resistant pathogen A. hydrophila was isolated, identified, and tentatively used in therapy. Ahy-yong1 possesses a head of approximately 66 nm in diameter and a short tail of approximately 26 nm in length and 32 nm in width. Its complete dsDNA genome is 43,374 bp with a G + C content of 59.4%, containing 52 predicted opening reading frames (ORFs). Taxonomic analysis indicated Ahy-yong1 as a new species of the Ahphunavirus genus of the Autographiviridae family of the Caudoviricetes class. Ahy-yong1 was active only against its indicator host strain among the 35 strains tested. It is stable at 30-40 °C and at pH 2-12. Aeromonas phage Ahy-yong1 revealed an effective biofilm removal capacity and an obvious protective effect in brocade carp (Cyprinus aka Koi). The average cumulative mortality for the brocade carp in the blank groups intraperitoneally injected with PBS was 1.7% ± 2.4%;for the control groups treated with A. hydrophila (108 CFU/fish) via intraperitoneal injection, it was 100.00%;and for the test group I, successively treated with A. hydrophila (108 CFU/fish) and Aeromonas phage Ahy-yong1 (107 PFU/fish) via intraperitoneal injection witha time interval of 2 hours, it was only 43.4% ± 4.7%. Furthermore, the cumulative mortality of the test group II, successively treated with Aeromonas phage Ahy-yong1 (107 PFU/fish) and A. hydrophila (108 CFU/fish), was only 20.0% ± 8.2%, and that of the test group III, simultaneously treated with Aeromonas phage Ahy-yong1 (107 PFU/fish) and A. hydrophila (108 CFU/fish), was only 30.0% ± 8.2%. The results demonstrated that phage Ahy-yong1 was very effective in the therapies against A. hydrophila A18, prophylaxis was more effective than rescue, and earlier treatment was better for the reduction of mortality. This study enriches knowledge about Aeromonas phages.

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.

Characterization of Three Novel Virulent Aeromonas Phages Provides Insights into the Diversity of the Autographiviridae Family.

In this study, we isolated and characterized three novel virulent Autographiviridae bacteriophages, vB_AspA_Bolek, vB_AspA_Lolek, and vB_AspA_Tola, which infect different Aeromonas strains. These three host–pathogen pairs were derived from the same sampling location—the arsenic-containing microbial mats of the Zloty Stok gold mine. Functional analysis showed they are psychrotolerant (4–25 °C), albeit with a much wider temperature range of propagation for the hosts (≤37 °C). Comparative genomic analyses revealed a high nucleotide and amino acid sequence similarity of vB_AspA_Bolek and vB_AspA_Lolek, with significant differences exclusively in the C-terminal region of their tail fibers, which might explain their host range discrimination. The protein-based phage network, together with a phylogenetic analysis of the marker proteins, allowed us to assign vB_AspA_Bolek and vB_AspA_Lolek to the Beijerinckvirinae and vB_AspA_Tola to the Colwellvirinae subfamilies, but as three novel species, due to their low nucleotide sequence coverage and identity with other known phage genomes. Global comparative analysis showed that the studied phages are also markedly different from most of the 24 Aeromonas autographiviruses known so far. Finally, this study provides in-depth insight into the diversity of the Autographiviridae phages and reveals genomic similarities between selected groups of this family as well as between autographiviruses and their relatives of other Caudoviricetes families.

Complete genome sequence analysis and phylogenetic classification of the novel Aeromonas phage AHP-1, a potential member of the genus Tequatrovirus.

Aeromonas phage AHP-1 was originally isolated from crucian carp (Carassius carassius) tissue. It was able to infect Aeromonas hydrophila and A. salmonicida. Genome sequence analysis revealed a 218,317-bp-long linear genome with an overall G + C content of 47.9%, 315 open reading frames (ORFs), and 25 tRNA sequences. Its genome was found to contain 67 unique ORFs (21.26%) that did not show any homology to previously characterized proteins. A comparative genome analysis suggested that its closest neighbors are unclassified phages belonging to the genus Tequatrovirus of the subfamily Tevenvirinae.

Effect of Antibiotics and Glycerol on Improving Bacteriophage Detection and Enumeration

Antibiotic-resistant bacteria are a growing issue in aquaculture, and phage therapy offers an alternative approach to the use of antibiotics. Phages for phage therapy can be isolated, identified and enumerated using a Double-Layer Agar (DLA) technique. While some phages form large and well-defined plaques that can be enumerated when plated with the DLA technique, some produce small and turbid plaques that are difficult to detect and count. A phage, Aeromonas phages-T65, was isolated from Aeromonas hydrophila, which formed small and turbid plaques making it hard to count. The use of antibiotics and chemicals have been suggested as a way to increase plaque size for easy detection and enumeration of phage plaques. To increase the plaque size of Aeromonas phages-T65, antibiotics and glycerol were added to the culture media, and tested at different concentrations. Application of penicillin-streptomycin and/or glycerol showed an increase in plaque size and the biggest improvement in plaque size were observed using either 40 U/μl penicillin-streptomycin or a 5 % glycerol in the top agar layer of the DLA technique. These findings suggest that such modifications could be implemented into the standard DLA overlay method to investigate new Aeromonas phages for the development of phage therapy against Aeromonas spp. in aquaculture.

Characterization and protective effects of lytic bacteriophage pAh6.2TG against a pathogenic multidrug-resistant Aeromonas hydrophila in Nile tilapia (Oreochromis niloticus).

Bacteriophage (phage) is considered as one of the alternatives to antibiotics and an environmentally friendly approach to tackle antimicrobial resistance (AMR) in aquaculture. Here, we reported isolation, morphology and genomic characterizations of a newly isolated lytic phage, designated pAh6.2TG. Host range and stability of pAh6.2TG in different environmental conditions, and protective efficacy against a pathogenic multidrug-resistant (MDR) Aeromonas hydrophila in Nile tilapia were subsequently evaluated. The results showed that pAh6.2TG is a member of the new family Chaseviridae which has genome size of 51,780bp, encoding 65 putative open reading frames (ORFs) and is most closely related to Aeromonas phage PVN02 (99.33% nucleotide identity). The pAh6.2TG was highly specific to A. hydrophila and infected 83.3% tested strains of MDR A. hydrophila (10 out of 12) with relative stability at pH 7-9, temperature 0-40°C and salinity 0-40 ppt. In experimental challenge, pAh6.2TG treatments significantly improved survivability of Nile tilapia exposed to a lethal dose of the pathogenic MDR A. hydrophila, with relative per cent survival (RPS) of 73.3% and 50% for phage multiplicity of infection (MOI) 1.0 and 0.1, respectively. Phage treatment significantly reduced the concentration of A. hydrophila in both water and fish body. Interestingly, the surviving fish from A. hydrophila challenged groups provoked specific antibody (IgM) against this bacterium. In summary, the findings suggested that the lytic phage pAh6.2TG is an effective alternative to antibiotics to control MDR A. hydrophila in tilapia and possibly other freshwater fish.

Metagenomic phage biodiversity: New insight into the tropical estuarine ecosystem process and microbial risk assessment

Viruses infecting unicellular organisms were shown to have a significant influence on aquatic ecosystem processes. Their role in biogeochemical cycles, primary production and microbial risk assessment in the tropical estuarine systems remain unresolved. The present study is the first approach of Next Generation Sequence (NGS) based viral metagenomic analysis of Cochin estuarine sediment. Viral particles were purified from 1.5 kg sediment samples collected from Cochin estuary, employing flocculation, and the purity was confirmed using epifluorescence microscopy. The flocculate was subjected to whole genome amplification using Illumina. Present NGS data provides information about the abundance of major bacteriophages, especially, cyanophages which play a significant role in ecosystem engineering and the biogeochemical cycle. In the present study, bacteriophages such as phages of Aeromonas, Bacillus, Salmonella, Vibrio, Burkholderia, Acanthocystis turfacea, Acidianus, Clostridium and Escherichia represented a major fraction. NGS result confirmed Aeromonas phages (19.78%) to be the most abundant communities present in the Cochin estuarine sediment. Cyanophages and phytoplankton attacking phage communities included A. turfacea virus, Chlorella virus, Acidianus filamentous virus, Ectocarpus siliculosus virus, Phaeocystis globosa virus, Chrysochromulina ericina virus, Emiliania huxleyi virus, Micromonas pusilla virus, Ostreococcus lucimarinus virus, Ostreococcus tauri virus, Synechococcus virus and Prochlorococcus virus. Unicellular photosynthetic Synechococcus and Prochlorococcus, abundant in oceans and estuaries, contribute to a substantial proportion of primary productivity, so the presence of these low-light adapted cyanobacteria attacking phages in estuarine sediments reveals that they are the key factor that controls the primary production and are also essential in preventing pathogenic bacterial population explosions. The data can be used as a road map for modelling efforts to link the role of viruses in determining carbon and nutrient availability in tropical estuarine ecosystems and the microbial risk assessment.

Molecular Characterization of Ahp2, a Lytic Bacteriophage of Aeromonas hydrophila.

Aeromonas hydrophila is an opportunistic pathogen that infects fish, amphibians, mammals, and humans. This study isolated a myophage, vB_AhyM_Ahp2 (Ahp2), that lytically infects A. hydrophila. We observed that 96% of the Ahp2 particles adsorbed to A. hydrophila within 18 min. Ahp2 also showed a latent period of 15 min with a burst size of 142 PFU/cell. This phage has a linear double-stranded DNA genome of 47,331 bp with a GC content of 57%. At least 20 Ahp2 proteins were detected by SDS-polyacrylamide gel electrophoresis; among them, a 40-kDa protein was predicted as the major capsid protein. Sequence analysis showed that Ahp2 has a genome organization closely related to a group of Aeromonas phages (13AhydR10RR, 14AhydR10RR, 85AhydR10RR, phage 3, 32 Asp37, 59.1), which infect Aeromonas hydrophila and Aeromonas salmonicida. The tail module encompassing ORF27-29 in the Ahp2 genome was present in all Aeromonas phages analyzed in this study and likely determines the host range of the virus. This study found that Ahp2 completely lyses A. hydrophila AH300206 in 3.5 h at a MOI of 0.0001 and does not lysogenize its host. Altogether, these findings show that Ahp2 is a lytic Aeromonas phage and could be a candidate for therapeutic phage cocktails.

A Novel Group of Promiscuous Podophages Infecting Diverse Gammaproteobacteria from River Communities Exhibits Dynamic Intergenus Host Adaptation.

Phages are generally described as species specific or even strain specific, implying an inherent limitation for some to be maintained and spread in diverse bacterial communities. Moreover, phage isolation and host range determination rarely consider the phage ecological context, likely biasing our notion on phage specificity. Here we isolated and characterized a novel group of six promiscuous phages, named Atoyac, existing in rivers and sewage by using a diverse collection of over 600 bacteria retrieved from the same environments as potential hosts. These podophages isolated from different regions in Mexico display a remarkably broad host range, infecting bacteria from six genera: Aeromonas, Pseudomonas, Yersinia, Hafnia, Escherichia, and Serratia Atoyac phage genomes are ∼42 kb long and highly similar to each other, but not to those currently available in genome and metagenome public databases. Detailed comparison of the phages' efficiency of plating (EOP) revealed variation among bacterial genera, implying a cost associated with infection of distant hosts, and between phages, despite their sequence similarity. We show, through experimental evolution in single or alternate hosts of different genera, that efficiency of plaque production is highly dynamic and tends toward optimization in hosts rendering low plaque formation. However, adaptation to distinct hosts differed between similar phages; whereas one phage optimized its EOP in all tested hosts, the other reduced plaque production in one host, suggesting that propagation in multiple bacteria may be key to maintain promiscuity in some viruses. Our study expands our knowledge of the virosphere and uncovers bacterium-phage interactions overlooked in natural systems.IMPORTANCE In natural environments, phages coexist and interact with a broad variety of bacteria, posing a conundrum for narrow-host-range phage maintenance in diverse communities. This context is rarely considered in the study of host-phage interactions, typically focused on narrow-host-range viruses and their infectivity in target bacteria isolated from sources distinct to where the phages were retrieved from. By studying phage-host interactions in bacteria and viruses isolated from river microbial communities, we show that novel phages with promiscuous host range encompassing multiple bacterial genera can be found in the environment. Assessment of hundreds of interactions in diverse hosts revealed that similar phages exhibit different infection efficiency and adaptation patterns. Understanding host range is fundamental in our knowledge of bacterium-phage interactions and their impact on microbial communities. The dynamic nature of phage promiscuity revealed in our study has implications in different aspects of phage research such as horizontal gene transfer or phage therapy.

Characterization of Novel Bacteriophage AhyVDH1 and Its Lytic Activity Against Aeromonas hydrophila.

Phage therapy is an alternative approach to overcome the problem of multidrug-resistant bacteria. Here, a novel bacteriophage AhyVDH1, which infects Aeromonas hydrophila 4572, was isolated and its morphology, one-step growth curve, lytic activity, stability under various conditions, and genome were investigated. Transmission electron microscopy revealed that AhyVDH1 has an icosahedral head 49nm in diameter and a contractile tail 127nm in length, suggesting that it belongs to the family Myoviridae. AhyVDH1 showed strong adsorption to the surface of A. hydrophila 4572 (90% in 10min). The latent period of AhyVDH1 was shown to be 50min, and the burst size was 274 plaque-forming unit/infected cell. AhyVDH1 was stable at 30°C for 1h and lost infectivity after20 min of heating at 60°C. Infectivity remained unaffected at pH 6-7 for 1h, while the bacteriophage was inactivated at pH < 4 or > 11. AhyVDH1 has a 39,175-bp genome, with a 58% G + C content and 59 open reading frames. BLAST analysis indicated that the genome sequence of phage AhyVDH1 was related to that of Aeromonas phage Ahp2. Both time and MOI-dependent in vitro A. hydrophila growth inhibition were observed with AhyVDH1.Re-growth of the host bacteria appeared about 12h after treatment, suggesting its potential therapeutic value in treating A. hydrophila infections, but phage cocktails should be developed.

Characterization and Genome Analysis of a Novel Marine Alteromonas Phage P24.

Although Alteromonas is ubiquitous in the marine environment, very little is known about Alteromonas phages, with only ten, thus far, being isolated and reported on. In this study, a novel double-stranded DNA phage, Alteromonas phage P24, which infects Alteromonas macleodii, was isolated from the coastal waters off Qingdao. Alteromonas phage P24 has a siphoviral morphology, with an icosahedral head, 61 ± 1nm in diameter, and a tail length of 105 ± 1nm. Alteromonas phage P24 contains lipids. It has an optimal temperature and pH for growth of 20℃ and 5-7, respectively. A one-step growth curve shows a latent period of 55min, a rise period of 65min, and an average burst size of approximately 147 virions per cell. Alteromonas phage P24 has the genome of 46,945bp with 43.80% GC content and 74 open reading frames (ORFs) without tRNA. The results of the phylogenetic tree, based on the mcp and terL genes, show that Alteromonas phage P24 is closely related to Aeromonas phage phiARM81ld. Meanwhile, phylogenetic analysis based on the whole genome of P24 indicates that it forms a unique viral sub-cluster within Siphoviridae. This study contributes to the understanding of the genomic characteristics and the virus-host interactions of Alteromonas phages.

Nine Novel Phages from a Plateau Lake in Southwest China: Insights into Aeromonas Phage Diversity.

Aeromonas species are common pathogens of fish and some of them can opportunistically cause infectious diseases in humans. The overuse of antibiotics has led to the emergence of bacterial drug-resistance. To date, only 51 complete genome sequences of Aeromonas phages are available in GenBank. Here, we report the isolation of nine Aeromonas phages from a plateau lake in China. The protein cluster, dot plot and ANI analyses were performed on all 60 currently sequenced Aeromonas phage genomes and classified into nine clusters and thirteen singletons. Among the nine isolated phages, the DNA-packaging strategy of cluster 2L372D (including 2L372D, 2L372X, 4L372D, 4L372XY) is unknown, while the other five phages use the headful (P22/Sf6) DNA-packaging strategy. Notably, the isolated phages with larger genomes conservatively encode auxiliary metabolism genes, DNA replication and metabolism genes, while in smaller phage genomes, recombination-related genes were conserved. Finally, we propose a new classification scheme for Aeromonas phages.

Complete genome sequences of Aeromonas and Pseudomonas phages as a supportive tool for development of antibacterial treatment in aquaculture

BackgroundAquaculture is the fastest growing sector of food production worldwide. However, one of the major reasons limiting its effectiveness are infectious diseases among aquatic organisms resulting in vast economic losses. Fighting such infections with chemotherapy is normally used as a rapid and effective treatment. The rise of antibiotic resistance, however, is limiting the efficacy of antibiotics and creates environmental and human safety concerns due to their massive application in the aquatic environment. Bacteriophages are an alternative solution that could be considered in order to protect fish against pathogens while minimizing the side-effects for the environment and humans. Bacteriophages kill bacteria via different mechanisms than antibiotics, and so fit nicely into the ‘novel mode of action’ concept desired for all new antibacterial agents.MethodsThe bacteriophages were isolated from sewage water and characterized by RFLP, spectrum of specificity, transmission electron microscopy (TEM) and sequencing (WGS). Bioinformatics analysis of genomic data enables an in-depth characterization of phages and the choice of phages. This allows an optimised choice of phage for therapy, excluding those with toxin genes, virulence factor genes, and genes responsible for lysogeny.ResultsIn this study, we isolated eleven new bacteriophages: seven infecting Aeromonas and four infecting Pseudomonas, which significantly increases the genomic information of Aeromonas and Pseudomonas phages. Bioinformatics analysis of genomic data, assessing the likelihood of these phages to enter the lysogenic cycle with experimental data on their specificity towards large number of bacterial field isolates representing different locations.ConclusionsFrom 11 newly isolated bacteriophages only 6 (25AhydR2PP, 50AhydR13PP, 60AhydR15PP, 22PfluR64PP, 67PfluR64PP, 71PfluR64PP) have a potential to be used in phage therapy due to confirmed lytic lifestyle and absence of virulence or resistance genes.

Characterization of a novel lytic myophage, phiA8-29, infecting Aeromonas strains.

Members of the bacterial genus Aeromonas are important aquatic pathogens that cause severe fish diseases. Here, we characterize a novel lytic phage, Aeromonas virus phiA8-29, isolated from the alimentary tract of a freshwater fish. Transmission electron microscopy showed that phiA8-29 has a long contractile tail and thus can be classified as a member of the family Myoviridae. The phage genome was identified as a double-stranded DNA molecule of 144,974 bp containing 185 open reading frames and nine tRNA-encoding genes. Comparative genomic analysis revealed that the phiA8-29 genome has little similarity to any of the currently sequenced Aeromonas phage genomes. Our data indicate that phiA8-29 is a novel lytic Myoviridae phage that does not belong to any of the known genera.