Isolation, Characterisation and Preservation of Bacteriophages for Use in Treatment of Aeromonas hydrophila Infection In Finfish

Abstract

Problems caused by multidrug resistant bacteria and the limited number of control strategies, highlight the need for alternative control approaches against bacterial infections in the aquaculture sector. Bacteriophages (phages) demonstrated promising results as therapeutic tools, for the effective control of bacterial diseases in aquaculture, as well as in other production animals, companion animals and humans. In the present study, a range of investigations were undertaken to assess suitability of using lytic phages in controlling Aeromonas hydrophila infection in fish aquaculture. A. hydrophila is a fish pathogen and responsible for economic damages to the aquaculture industry. The main focus of this study was to develop and use a suitable model to test the efficacy of a phage therapy by addressing the concerns associated with the potential limitations of the therapy, such as required doses and delivery methods, characterisation at the genomic level, storage methods, and the phage therapy effect, potential immune response, and effects on fish in response to multiple phage treatments. For the isolation of suitable A. hydrophila phages, wastewater was used as a source and ten different A. hydrophila strains were used as hosts to culture and screen potential phages with lytic ability. Before use in isolation, the bacterial strains were confirmed for any prophage induction using Mitomycin C assay. The water samples were filtered to retain only virus particles and enriched with A. hydrophila strains, filtered again to remove the bacterial hosts and then subjected to a series of plaque assays to screen potential lytic phages. The morphology of the isolated phages was characterised by Transmission Electron Microscopy (TEM); genetic organisation was determined by enzymatic digestions of their nucleic acids and lytic ability was determined by growth pattern while growing with their respective host bacteria. Four selected phages were subjected to whole genome sequencing using Illumina technologies with a view to determining their phylogeny, adaptive evolution, biodiversity, potential virulence and presence of any toxin genes. The isolated and selected phages were then subjected to a study to determine their shelf lives under long-term storage conditions as a solution and in a freeze-dried form. To store as a solution, effect of different concentrations of sucrose and trehalose on the bacteriophage titre were compared with no sugar treatments under 4 °C, -20 °C and -80 °C for 56 weeks. To store in a lyophilised form, bacteriophage solutions were prepared with and without sugars (sucrose and trehalose) and then freeze dried and stored at 4 ℃ and room temperature for 44 weeks. For both trials, bacteriophage titre was monitored weekly for first 8 weeks and then monthly until the end of the experiments. Any changes in phage morphology after lyophilisation was determined by TEM. The therapeutic potential of isolated phages was assessed in fish trials with a native Australian fish, silver perch (Bidyanus bidyanus) against one strain of A. hydrophila. A cocktail of four selected phages was prepared. Different phage doses were determined based on different phage titres to be tested on live fish models infected with A. hydrophila to understand the effects of environmental conditions and dose response of the therapeutic mode. The fish infection trial was conducted in two different phases – preliminary pilot trial and actual infection challenge trial. The preliminary pilot trial utilised 15 fish intraperitoneal injected with different quantities of A. hydrophila and a control group with phosphate buffered saline (PBS). After 6 days, the infected fish were euthanized and organs were collected for bacterial load determination. In the actual infection challenge trial, 144 fish were employed in three groups – control (no treatment, only PBS); A. hydrophila infected fish treated with different doses of bacteriophage cocktails; and uninfected fish treated with different doses of bacteriophage cocktails. At the end of the trial, bacterial load was counted from spleen and kidney and histopathology conducted on various fish organs. Fish blood was analysed for presence/absence of A. hydrophila and to determine lysozyme activity and IgG levels as indicators of immunological response to bacteria or the bacteriophage. Screening for potential A. hydrophila phages with lytic ability resulted in the selection of four phages, coded as Ah6_G, Ah9_PP, Ah10_Se and Ah10_S. The isolated phages demonstrated complete lysis against their respective hosts and contained non-contractile tails with icosahedral heads belonging to Siphoviridae family (Ah6_G, Ah10_Se and Ah10_S), and icosahedral heads with short tail belonging to Podoviridae family (Ah9_PP). All the isolated phages contained dsDNA of > 10 Kb as their genetic materials. The whole genome analysis revealed that all these four phages are closely related to Ahp1 podovirus in terms of their genetic similarity. The whole genome sequencing of the selected phages confirmed that all the isolates contained typical phage protein coding genes, ds DNA and smaller sizes of genomes of Ah6_G (circular), Ah10_Se (linear) and Ah10_S (linear) (47-61 Kb) than other Siphoviridae phages, However, size of the genome of Ah9_PP (41 Kb, circular) was similar to other Podoviridae phages. Two of the phages (Ah10_Se and Ah10_S) were identified as novel linear dsDNA phages with protein coding sequences having no similarities found in the existing phage databases. The phage shelf life study revealed that solutions of the four isolated phages could be preserved without any sugar stabilizers for 56 weeks at both 4 ℃ and -80 ℃ with no significant reduction in their titre. However, -20 ℃ was found to be less effective temperature in maintaining shelf lives of phages Ah6_G and Ah9_PP. Interestingly, the shelf life of these two phages at -20 ℃ were significantly improved when sucrose or trehalose added, sucrose being the better protectant. Nevertheless, the sugars did not have any adverse effects on their shelf lives over the experimental periods. When lyophilised, a small reduction in titre of Ah10_Se and Ah10_S was observed which was improved by adding sucrose and trehalose. Exclusion of sugars during lyophilisation caused disruption of heads and tails of the viruses. In the presence of sucrose or trehalose, the lyophilised phages were more stable when stored at 4 °C compared to room temperature. Nontheless, the presence of sucrose or trehalose provided better stability to the lyophilised phages at either temperatures. However, storing the phages in solution was proved to be better than lyophilisation over the experimental periods. In the fish trial to assess therapeutic potential of isolated phages, a phage cocktail was used to treat A. hydrophila infections in silver perch fish model. Unfortunately, when purchased the fish had already been exposed to A. hydrophila. A medium dose of phage cocktail significantly reduced the background A. hydrophila count in the kidney and spleen of the fish group that did not receive intraperitoneal bacterial inoculation. When the fish were deliberately infected with exogenous A. hydrophila on top of the existing colonisation with Aeromonas sp., there were no significant differences in bacterial counts when challenged with any of the doses of phages. No significant immune response was observed during the challenge trial. The comparative histopathology study of different tissues of the experimental fish revealed very few significant changes that are consistently associated with A. hydrophila infection in teleost fish. This thesis provides new information on the properties of phages able to kill A. hydrophila and that have potential for use as treatments in controlling A. hydrophila infection in fish. Further studies are required to conduct larger scale fish trials involving different species of fish to expand, validate and implement the knowledge and approaches generated in this study.