dc.description.abstract
Antibacterial activity of marine sea cucumber and oyster against Staphylococcus aureus and Vibrio
Oceans and seas are home to a large proportion of all life on Earth, with 71% of the Earth's surface being ocean water, while only 2.5% is drinkable freshwater. Oceanic plankton, algae, and certain bacteria contribute significantly to this large proportion. Marine habitats are divided into coastal and open ocean habitats, with coastal habitats occupying only seven per cent of the ocean surface. The organisms studied range from microscopic phytoplankton and zooplankton to whales in the open ocean. Invertebrates are an important part of marine life, providing food, medicine, and raw materials. Advancement of invertebrate-derived compounds is a therapeutic prospect for biomedical discovery. Marine natural product drug discovery efforts have yielded nine approved drugs and 12 currently under clinical trial, largely from collections of marine invertebrates from shallow-water tropical ecosystems. The global fish production industry has a significant impact on national economies, providing 17% of the animal protein consumed by the global population, and providing health benefits. Aquaculture is a significant contributor to the global food fishery industry.
Sea cucumber is a marine invertebrates widely consumed in East Asia due to its multiple biological and pharmacological properties, including anticancer, anti-angiogenic, anticoagulant, anti-inflammatory, anti-hypertension, antimicrobial, antithrombotic, antioxidant, and antitumor properties. The Persian Gulf is a biodiverse ecosystem with Holothuria leucospilota being the most commonly harvested sea cucumber. However, only 20 articles have investigated the Persian Gulf's Holothurian species. Previous investigations have shown that high concentrations of Holothurian compounds have a weak antibacterial effect. Sea cucumber protein hydrolysates are a healthy and reliable alternative to artificial food preservatives. Staphylococcus aureus is one of the most prevalent pathogens responsible for food poisoning, and the emergence of antimicrobial resistance in S. aureus is a public health concern.
In chapter II, I studied by using extraction of bioactive compounds from sea cucumber Holothuria leucospilota revealed antibacterial effects on MRSA and enterotoxin-producing Staphylococcus aureus strains (SEASA, SEBSA) were determined. Three methods were used to determine antimicrobial activity: disc diffusion experiments, the minimum bactericidal concentration (MBC), and the minimum inhibitory concentration (MIC). The results indicate that methanol and chloroform extracts inhibit the growth of all tested isolates at MIC concentrations as high as 100 mg/ml. In concentrations of approximately 100 mg/ml, the chloroform extract exhibited bactericidal activity against SEBSA. In concentrations below 100 mg/ml, the extract is also bactericidal against MRSA and SEBSA. Methanol extract demonstrated the greatest antibacterial activity. Therefore, sea cucumber extract is a promising candidate for the discovery of new antimicrobials; however, extensive research is required to isolate and identify the active components of Holothuria leucospilota from the Persian Gulf. I found Sea cucumber extracts can considered to possess antimicrobial properties. My results support the idea that the sea cucumber extracts are considered a healthy and dependable alternative to artificial food preservatives is illustrative of their antimicrobial activity.
Aquaculture contributes to global fish production and health benefits.Vibriosis is a potentially fatal infection caused by Vibrio species, which are abundant in marine and estuarine ecosystems and can cause human cutaneous infections and gastrointestinal disorders. Vibrio spp is increasing due to climate change, and Pacific Oyster Crassostrea gigas is an appropriate model for the study of Vibrio and host interactions in disease dynamics. Investigation of interactions between oysters and vibrio.spp is essential to prevent bacterial diseases and mortality in aquaculture. Fluorescence in situ Hybridization FISH was used to visualize diversity of v.Kanaloae in different tissues.
In chapter III, investigated on the C. gigas oysters from Sylt Island were exposed108 V. kanaloae strains T02 and S12 in the summer of 2020 .Consequently, different oyster organs were analyzed by the culture-independent molecular technique, Fluorescence in situ Hybridization (FISH) to rapidly visualize the diversity of v.Kanaloae in different tissues of the oyster on the 3rd day post-infection due to the fact that cellular immune parameters peaked on day 3 demonstrating primarily a response to the immune challenge. Consequently, our findings offer a novel perspective on invasion efficacy. During an investigation into its in vivo distribution, the digestive organs, gills, and muscles were found to contain Vibrio kanaloae.
Aquaculture production including fish, oysters, molluscs exceeded 177.8 million metric tonnes in 2019. Vibrio splendidus is affecting global production of C. gigas oysters. Oysters have an integrated, highly complex innate immune system to identify and eliminate invaders, with both innate and adaptive immunity. The genome of the Pacific oyster C. Gigas has revealed massive expansion and functional divergence of innate immune genes. Immune priming in enhances the immune response upon subsequent exposure to V. splendidus. Immune priming involves two mechanistic models: a biphasic response followed by an extinction phase, and a unique response initiated after priming . Immunostimulants can increase the resistance of aquatic organisms to infectious diseases by enhancing their defense mechanisms.
In chapter IV, I investigated on Pacific oysters (Crassostrea gigas) primed with 104 Vibrio splendidus exhibited stronger immune responses when challenged with 107 V. splendidus. In addition, we inoculated the oysters against Vibrio by exposing them to sympatric and allopatric V. splendidus strains in order to evaluate their resistance in vivo and determine if this colonization increases virulence and survival. Oysters that had been cross-protected against the sympatric strain of V. splendidus survived a secondary challenge better. In addition, we examined the priming of the bacterium Vibrio by exposure to colonized oysters in order to determine their resistance to haemolymph in vivo experiment. We analyzed the immunological responses of oysters to determine if colonization leads to an increase in mortality. In the oyster model, the selected isolates demonstrated increased survival in both the primary and secondary challenges, as well as in vitro protection against V. splendidus. Selective populations of oysters resistant to V. splendidus exhibited an increase in in vivo survival, an inhibitory effect on V. splendidus in vitro during a secondary challenge, and cross-protection against V. splendidus in vitro examination.
In this thesis, I investigated hypotheses explaining the Sea Cucumber H. leucospilota has antibacterial effects against S. aureus strains MRSA, SEASA, and SEBSA, but further research is needed. FISH is a valuable and rapid technique for detecting and quantifying V. kanaloae's living forms. Using the developed method, viable V. kanaloae in oysters can be identified. The Vib-16S-1 instrument can be used to determine the pathogenic species of Vibrio that is most prevalent. Due to the direct visualization of bacteria, the detection time can be significantly reduced compared to culture-based methods. Immunopriming was shown to stimulate oyster humoral immune responses against both sympatric and allopatric strains of V.splendidus in order to improve specific memory and adaptive immunity in oysters. which could be used for oral vaccine programs to prevent aquaculture disease outbreaks. Further investigation is required to determine the prospective mechanism responsible for the observed changes.
Objective
In my dissertation, I conducted an in-depth investigation into microbial interactions in aquatic organisms and their implications for seafood safety, disease dynamics, and biocontrol strategies. The study delves into the intricate realm of microbial interactions, with a specific focus on the sea cucumber Holothuria leucospilota and the Pacific oyster Magalana gigas. Through a series of interconnected chapters, the research endeavors to establish a thorough understanding of these interactions, offering valuable insights into seafood safety, the dynamics of diseases, and the potential development of biocontrol strategies.
Sea cucumber Holothuria leucospilota Extracts
This chapter explores the antimicrobial potential of extracts obtained from Holothuria leucospilota, emphasizing their effectiveness against Staphylococcus aureus, including methicillin-resistant strains and enterotoxin producing Staphylococcus aureus strains (SEASA, SEBSA. The primary objective was to identify bioactive compounds within the sea cucumber extracts possessing antibacterial properties. By doing so, this chapter lays the groundwork for investigating natural antimicrobials in marine organisms, positioning the sea cucumber as a promising source for the discovery of novel antimicrobial agents and a particular emphasis on their suitability as substitutes for synthetic food preservatives.
Vibrio Dynamics in Oysters
Chapter III examined the relationship between Pacific oysters (Crassostrea gigas) and Vibrio species, particularly Vibrio kanaloae, in the context of disease dynamics. The technique of molecular fluorescent in situ hybridization (FISH) was used to quickly determine the range of bacteria present in Crassostrea gigas. This allowed for important observations on the metabolic activity and distribution of Vibrio strains throughout the tissues of the oysters. This chapter establishes the fundamental concepts necessary to comprehend the dynamics between hosts and pathogens, thereby creating a foundation for investigating the influence of microbial interactions on the health of oysters.
Immune Priming in Oysters
In this chapter, our main goal is to investigate the specificity of immune priming responses in Pacific oysters (Magalana gigas) as a potential strategy for biocontrol against pathogenic Vibrio strains, particularly Vibrio splendidus, with a focus on mitigating high summer mortalities in aquaculture. We explore the nuances of immune priming by exposing oysters to sympatric and allopatric V. splendidus strains, assessing the inhibitory effects of haemolymph plasma, and examining cross-protection against lethal Vibrio challenges. The research sheds light on the correlation between strain origin and the effectiveness of immune priming, emphasizing the potential of matching local strains for optimal protection. Ultimately, the findings aim to contribute to the development of targeted biocontrol measures to safeguard oyster production and address economic implications in the food industry.
Conclusion
Expanding on the knowledge acquired from Chapters II, III, and Chapter IV, this section explores the immunological priming responses demonstrated by Pacific oysters, namely Magalana gigas, when exposed to pathogenic Vibrio strains. The objective is to examine the degree of immunological priming specificity by studying closely similar strains of Vibrio splendidus. This chapter attempts to investigate the possibility of immune priming as a biocontrol technique against high summer mortalities in oyster populations by analyzing the inhibitory effects of oyster haemolymph on Vibrio growth.Collectively, these chapters establish a coherent story that covers the discovery of natural antimicrobials in marine organisms (Chapter II), the comprehension of disease patterns in oysters (Chapter III), and the investigation of immune priming as a possible approach to control Vibrio-induced mortality in aquaculture (Chapter IV). This dissertation enhances our comprehension of microbial interactions in aquatic habitats, which has significant implications for ensuring seafood safety and implementing sustainable management practices in aquaculture systems.
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