The causative agent of Motile Aeromonas Septicaemia (MAS) in fish is Aeromonas hydrophila. This gram negative motile bacteria normally inhabits in aquatic ecosystem and is an opportunistic pathogen and zoonotic in nature causing gastroenteritis, septicaemia in humans. In aquaculture, this bacterium can lead to production losses and economic distress to the farmers. Antibiotics have been the most prevalent control option for MAS in both hatcheries and farms [16]. But the major problem with these antimicrobial agents is that, they not only kill target bacteria but might harm host’s natural habitat and water quality parameters. In addition this has also led to a new set of problems in the form of antibiotic resistance which is one of the major concerns globally. A good example of this can be found in recent research works that, with the frequent use of antibacterial drugs in aquaculture, multidrug resistance (MDR) of Aeromonas hydrophila strains are increasing [6, 18]. In another work, the researchers analysed the percentage frequency of antibiotic resistant A. hydrophila strains from fish and prawns and found that they were resistant to methicillin, rifampicin, bacitracin, and novobiocin [24]. In addition to this, 43 Aeromonas strains isolated by Hossain et al. [11] from 46 zebra fish were resistant to four antibiotics. These MDR strains can reproduce and pass its resistance on, creating many more antibiotic resistant bacteria, which is a major threat to aquaculture species. Moreover, A. hydrophila forms bio-film on host tissues and these biofilm-embedded bacteria are one of the major causes of diseases in aquaculture [4]. This is because of the fact that, bacteria within a biofilm structure provides a reduced penetration of antibacterial compounds into the biofilm, making them more resistant to antibiotics [9]. Fishes use both specific and non-specific immunity mechanisms to protect themselves against these pathogens, and hence researchers have focussed their attention towards alternative treatment methods like vaccination, probiotics, immunostimulants and phage therapy [25]. Phages or bacteriophages are viruses that infect and replicate within bacteria. They are highly specific towards host at strain level. This is an advantage as they can be utilised to control the target bacterium without damaging the ecosystem microflora. Hence bacteriophages can be an excellent option to treat bacterial infections and has been used successfully in treating diseases in aquaculture. In an earlier work, Phage Biotech Ltd. in Israel, phage treatment was used in aquaculture to treat infections by Vibrio harveyi in shrimp [10]. This introduced a new branch in the field of aquaculture diseases treatment i.e., phage therapy and has been used in treating aquatic diseases in different works [12, 13, 20]. To understand the characters of bacteriophages and to have a better knowledge about its efficacy as a treatment option, whole genome sequencing is necessary before suggesting its implementation in aquaculture sector.
In the present study novel bacteriophage, named Aeromonas phage GomatiRiver_11 (G11) has been sequenced for the first time.
Phage genomic DNA extraction was carried as previously described by Su et al [23]. Phages were isolated from water samples from various ponds and river of all over Tripura (a North Eastern state of India) against A. hydrophila and they were purified following the methods described by Jun et al [14]. The phage sample was taken from purified sample, added RNase and incubated at 37°C for 30 min. Again the mixture was incubated at 30°C for 5 min after adding 2M ZnCl2 at a ratio of 1:50. After this the incubation mixture was centrifuged (4000g for 5 min) and the pellet was collected. Then to this proteinase K [Final Concentration 100 mg/ml] and Tans buffer was added followed by an incubation at 65°C for 10 min. After incubation equal volume of Phenol:Chloroform:Isoamyl alcohol (25:24:1) was added and centrifuged (4000g for 5 min). The aqueous phase was collected. Equal volume of Isopropanol was added and centrifuged (4000x g for 10 min.). Finally the DNA pellet was washed with 70% ethanol and suspended in TE buffer.
The phage DNA samples were taken for purification using AMPureXP beads (Beckman Coulter, USA) to remove the possibilities of contaminants. The quality and quantity of the purified DNA was checked on NanoDrop followed by 4200 TapeStation system (Agilent Technologies, USA) using gDNA Screen Tape (Agilent Technologies, USA). Then the paired-end sequencing libraries were prepared from the DNA sample using Illumina TruSeq Nano DNA Library Prep Kit (Illumina, USA) following the manufacturer’sprotocol. Initially, 200 ng of DNA was fragmented by Covaris M220 (Covaris, UK) to generate a mean fragment size of 350bp. As Covaris shearing generates dsDNA fragments with 3' or 5' overhangs, the fragments were then subjected to end-repair by NEBNext® End Repair Module (New England Biolabs, USA). The ligated products were size selected using AMPure XP beads (Beckman Coulter, USA). The size-selected product was PCR amplified with the index primer as described in the kit protocol. The PCR enriched libraries were analyzed on 4200 TapeStation system (Agilent Technologies, USA) using high sensitivity D1000 Screen Tape (Agilent Technologies, USA) as per manufacturer instructions. The paired-end (PE) Illumina libraries were loaded onto NextSeq500 for cluster generation and sequencing using 2 x 150 bp chemistry.
The sequenced raw data was processed to obtain high quality clean reads using Trimmomatic v0.39 [3] to remove adapter sequences and ambiguous reads. The high quality reads were aligned to the reference genomes of Aeromonas phage and genomes downloaded from NCBI sequence using BWA MEM v0.7.17 [17]. The mapped reads were assembled into scaffolds using SPAdes assembler v3.15.5 [21]. The scaffolds were subjected to homology search using BlastN against NCBI “NT” db and reference genomes of Aeromonas phage. The topmost phage hit(s) were selected for the downstream genome finishing using GFinisher v1.4 [8]. The scaffolds annotated as phages were taken for PHASTER (PHAge Search Tool Enhanced Release) [1]. Further gene prediction was executed using Prokka v1.14.5 [22]. The workflow of our bioinformatics pipeline has been shown in Fig 1. In addition to this for Transmission Electron Microscopy, ultracentrifugation at 24,500g at a temperature of 40C for 3 h (Hitachi, Himac CS150GXII, Japan) was done to get purified phages with high titre (1012-1014 PFU/ml). This was followed by washing twice with 0.1 M Ammonium acetate. 10 ul of washed phage was put on the carbon coated 400-mesh TEM copper grid (NisshinME, Tokyo, Japan) adding 2% uranyl acetate for 5 minutes. Then the samples were examined by TEM (Transmission electron microscopy) under 160,000X magnification (Technai G2 T12 BioTwin, TEM, Hillsboro, USA) and run at 120KV at IISC, Bengaluru, India.. The phylogenetic analysis of the sequenced phage genome with related phage genomes was done by Phylogeny.fr through PhyML method [5].
The phage genome is 10,390 bp in length with GC content of 44.1%. A total of 9 ORFs were identified. BLASTn analysis of the complete genome sequence showed the best match with the podovirus Aeromonas phage Asfd_1 (MK577502.1), with 85.83% identity (ID) and 99% query coverage (QC). There are no tRNAs in this phage genome. In an earlier study it has been discussed that, phages without tRNAs are significantly shorter than those with tRNAs [2]. Moreover, there are very less or no tRNAs in temperate phages [2]. This is why in the present study the phage genome length was around 10 kb. Out of the total 3.26 million PE reads, 0.78 million were mapped to the reference genome of Aeromonas phages. The PHASTER analysis revealed that the sequenced phage genome is complete i.e. intact (score > 90). The largest gene was found to be tail sheath monomer or tail protein and smallest being pro-head core protein with a length of 1991 and 245 bp, respectively. The phage genome annotation has been shown in Fig 2.
The TEM analysis showed that the bacteriophage has a icosahedral head and longer tail of 73.6 ± 14.8 nm long and 47.0 ± 9.5 nm wide and 209.2 ± 12.7 nm long and 17.6 ± 1.8 nm wide, respectively (Fig. 3). This range falls under the characteristic features of family Myoviridae according to ICTV- International Committee on Taxonomy of Viruses [15]. So the identified phage family is Myoviridae. The phylogenetic analysis showed that the phage genome is closely related to T4-like phages of the family Myoviridae and shared same clade with Aeromonas phage 44RR2.8t (Fig. 4).
The terminase large subunit gene characterized in the present study, is important for DNA packaging in bacteriophage. It acts as an ATP driven molecular motor necessary for viral DNA translocation into empty capsids and as an endonuclease that cuts the viral genome from the concetamer to initiate and to end the packaging response. The tail jacket monomer in the sequenced phage genome facilitates veritably high viral infection effectiveness and its compression ejects the contagion DNA into the host cytoplasm. The tail fiber in the phage genome canons for a protein, which is necessary for specific recognition of bacterial shells during the first step of viral infection [19]. The phage tail is attached to the portal whirlpool of the head. The head part protects the phage genome and made up of the major core protein, gp22, the minor core proteins, gpalt, a serine- type protease, gp21, and other internal proteins [7].
This newly identified phage in the present study will help in controlling A. hydrophila in aquaculture and further research.