Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmaceutical Sciences

First Advisor

David Rowley

Abstract

Aquaculture is a multi-billion dollar industry worldwide. The United States is a significant consumer to both fresh and marine aquaculture products. Aquaculture sales in Rhode Island have dramatically increased in the last 20 years. In Rhode Island nearly 4.3 million oysters were produced via aquaculture in 2012. Currently, hatcheries and nurseries in the United States produce large amounts of a variety of species of oysters, clams and scallops. Oysters are filter feeders and are exposed to many microbes in the hatchery. Infectious diseases from bacterial pathogens in the hatcheries can have serious impacts on production. Vibrio species are often responsible for vibriosis disease outbreaks in bivalve larviculture hatcheries worldwide. Another prevalent disease observed in oyster nurseries in the Northeastern US is Juvenile Oyster Disease (JOD).
Probiotic agents are promising tools to reduce the risks of disease outbreaks in aquaculture facilities. Two marine bacteria, Bacillus pumilus RI0695 and Phaeobacter gallaeciensis S4, were previously reported to provide significant protection of the Eastern oyster larvae Crassostreae virginica when challenged with the shellfish pathogen Vibrio tubiashii. The goals of my dissertation research were to isolate and identify the antibiotic(s) secreted by Phaeobacter gallaeciensis S4, to chemically examine their mechanisms of action for probiotic activity, and to create probiotic formulations of Bacillus pumilus RI0695 and of Phaeobacter gallaeciensis S4 for delivery in shellfish larviculture facilities.
Chapter 2 describes the isolation and identification of the antibiotic tropodithietic acid (TDA) from Phaeobacter gallaeciensis S4. Genes tdaA, tdaB, clpX and rpoE were previously found to be necessary for the biosynthesis of TDA in Silicibacter sp. TM104 (Geng, Bruhn et al. 2008, Karim, Zhao et al. 2013). Gene exoP is responsible for the exopolysaccharide biosynthesis (Zhao 2014). Collaborative work suggests that TDA contributes to the probiotic activity of P. gallaeciensis S4 but that antibiotic production is not the sole mechanism of action. The basis for this finding was biological and chemical analysis of S4 wild-type and genetic mutant strains producing different levels of TDA (tdaA-, tdaB-, tdbD-, clpX, rpoE-, exoP-, and complement strains rpoE-, exoP-) by high pressure liquid chromatography (HPLC) and ultra high pressure liquid chromatography (UHPLC). HPLC analysis of culture extracts from the tdaA-, tdaB- and tdbD- mutants confirmed loss of TDA production as compared to S4 wild type. Additional genetic mutant strains, clpX, rpoE-, exoP-, and complement strains rpoE- and exoP- were created by insertional mutagenesis to further explore the role of TDA and mechanisms regulating its production. UHPLC analysis of clpX stationary phase culture extracts confirmed the lost production of TDA when compared to a TDA standard. UHPLC analysis of complement strains clpX demonstrated that TDA was present compared to a TDA standard. The exoP- mutant produced TDA similar to the wild-type strain. Mutant strains that lack the production of TDA had less protection of Eastern oyster larvae C. virginica to bacterial challenge than the wild type or the genetic mutant strain that produced similar TDA concentrations as the wild type. This research determined that TDA was necessary for optimal probiotic activity.
Chapter 3 describes efforts to create a stable formulation of B. pumilus RI0695 for delivery at shellfish hatcheries. Currently there are no commercially available probiotics for shellfish aquaculture. Granulation is robust, cost effective, and simple proven method of formulation. A granular probiotic formulation of B. pumilus RI0695 was created by extruding dried B. pumilus RI0695 cells through three particle size sieves (40s, 80s, and 325s). Three granule sizes of 420 μ, 177 μ and 43 μ were successfully created. Granular (177 μ and 43 μ) formulations stored for 29 weeks and 22 weeks at room temperature (RT) were able to reduce mortality in C. virginica larvae and seed, respectively, when challenged with V. tubiashii. This study suggests the 43 μ granule formulation of B. pumilus RI0695 is a good candidate for commercial use in shellfish hatcheries.
In Chapter 4, a study is presented showing an effort to create a lyophilized probiotic formulation of P. gallaeciensis S4 that provides reduced mortality of C. virginica larvae when exposed to the shellfish pathogen V. tubiashii RE22. Several lyophilized formulations were prepared using varying amounts of two cryoprotectants at two growth stage phases of the bacterium. The two best formulations used log phase cells lyophilized with either 30% or and 40% mannitol as a cryoprotectant. The cell viabilities of the two formulations were measured under various storage conditions (27 °C, 4 °C, 30 °C, and 30 °C with 75% humidity) over a 5-week period. S4 formulations (30-M and 40-M) were tested at 1 week for probiotic protection of C. virginica seed against V. tubiashii infection. Unfortunately, the lyophilization process and storage significantly decreased the cell viability of both formulations (30-M and 40-M). Further, there was no protection of the larvae when pre-exposed to either formulation. Based on the in vivo results, a liquid P. gallaeciensis S4 formulation under starvation conditions in NSS medium was prepared. The liquid formulation maintained a cell viability of 108 CFU/mL over 8 weeks. Further research should be done to evaluate these formulations in a hatchery study. More research must be done to refine the formulation processes for commercial scale up.

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