Wright, Emily (2010) Bacteriophages and bacteriocins: agents for biocontrol of the fish pathogen Streptococcus iniae. Masters (Research) thesis, James Cook University.

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Abstract

Streptococcus iniae is one of the most widespread and costly pathogens in fish aquaculture. Bacteriophages (phages) and bacteriocins may be the optimal new therapeutic agents for S. iniae that lack many of the problems associated with current control measures. This study aims to discover and characterise phages and bacteriocins with activity against S. iniae.

Cross spotting assays and mitomycin C inductions were conducted to screen for prophage within the JCU library of 48 S. iniae isolates. Phages were induced with 150-350 ng ml-1 mitomycin C, isolated through filtration and concentrated by ultracentrifugation. Phages were characterized by plaque assays on S. iniae, transmission electron microscope analysis, and gel electrophoresis of EcoRI digested phage DNA. Mitomycin C inductions revealed 20.83% of S. iniae isolates show growth curves indicative of lysogeny. Plaque assays confirmed inducible prophages in 14.58% of isolates. Each of the induced phages displayed distinctive host ranges within the S. iniae library. Four phages, vB_SinS-44, vB_SinS-45, vB_SinS-46 and vB_SinS-48 lysed over 78% of isolates, and were highly concentrated. Phage vB_SinS-45 was extracted at the highest concentration of 4.2 × 1011 PFU ml-1 following ultracentrifugation. Transmission electron microscope analysis revealed phage particles from these four samples that exhibited long, non-contractile tails and isometric heads consistent with morphology of Siphoviridae. Restriction digests of phage DNA revealed two distinct cutting patterns, indicating similarity between phages in bacteria isolated from different hosts and geographic regions. The genome sizes estimated from the restriction digest are 28.5 kb and 66 kb for vB_Sin-45 and vB_Sin-46, respectively. The phages isolated and characterized in this study are the first described lysogenic phages associated with S. iniae, and are excellent candidates for genetic modification for use in therapy, prevention or research of S. iniae infections in fish.

To isolate lytic phages, environmental samples were collected from fish and prawn farms in Queensland. Numerous methods to expose S. iniae lytic phages were trialed, including enrichments in various sets of S. iniae isolates, filter-free isolation techniques to minimize damage to phages, and concentration of samples using ultracentrifugation and polyethylene glycol before and after enrichment processes. No lytic phages specific for S. iniae were successfully isolated in this study. Spatial and seasonal distribution of the host bacteria, as well as farm vaccination programs may have prevented the collection of phages from environmental samples. Lytic phages will likely be found on farms experiencing current outbreaks of S. iniae, none of which occurred during this study.

During the course of the project, a bacteriocin-like inhibitory substance (BLIS) with strong antibacterial activity against S. iniae was discovered. This BLIS, found to be produced by Lactococcus lactis ssp. lactis, was characterized through antagonism assays on the S. iniae library, plasmid curing tests, protein isolation, concentration, and SDS-PAGE analysis. The kinetics of production, effects on cell viability, heat, pH and enzyme tolerance of the BLIS were also determined. The BLIS was found to be a protein bacteriocin, termed BacL49, that is heat and pH stable (100°C for 60 min, pH 2.5-9.5), and sensitive to proteinase K, _-chymotrypsin, trypsin and papain. BacL49 active proteins were found to be 5 kDa and 54 kDa in size. The bacteriocin appeared not to be plasmid regulated. BacL49 is produced at the end of the log phase and antagonistic activity decreases following early stationary phase. The bacteriocin displays antagonism against 93.75% of S.iniae isolates. Though BacL49 is susceptible to trypsin, unlike most forms of nisin, sequencing of the protein needs to be completed before confirming that this bacteriocin is unique. BacL49 should be considered as a therapeutic agent against S. iniae, due to its heat and pH stability and bactericidal activity against the majority of S. iniae strains. BacL49 has the potential to be delivered to fish as a purified peptide or in the form of a live probiotic as L. lactis ssp. lactis L49.

Phage therapy remains an excellent potential tool for control of S. iniae in aquaculture, despite the inability of this study to isolate a lytic phage against the bacterium. Further study, in vivo trials, and manipulation of both the bacteriocin and phages isolated in this study could produce novel, effective biocontrol agents in the fight against S. iniae.

Item ID:	11782
Item Type:	Thesis (Masters (Research))
Keywords:	fish diseases, aquaculture, bacteriophages, bacteriocins, streptococcal infections, Streptococcus iniae, fish pathogens, phage therapy, protein bacteriocin BacL49, biological control
Date Deposited:	19 Aug 2010 05:37
FoR Codes:	07 AGRICULTURAL AND VETERINARY SCIENCES > 0704 Fisheries Sciences > 070404 Fish Pests and Diseases @ 50% 06 BIOLOGICAL SCIENCES > 0605 Microbiology > 060501 Bacteriology @ 50%
SEO Codes:	83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8301 Fisheries - Aquaculture > 830199 Fisheries - Aquaculture not elsewhere classified @ 100%
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