POLYPHASIC ANALYSES ON THE NATURAL ECOLOGY OF HUMAN PATHOGENS

The focus of research concerning human pathogens has been primarily centered on virulence in the host, transmission between hosts, and treatment of the subsequent infections. Justifiably, our well-being relies on such research but it has erroneously resulted in the assumption that the role of these microbial pathogens is to infect and reproduce within or on our bodies and then pass to another human to follow this same cycle ad infinitum. Although this does represent a true optional lifestyle for many pathogens, it must be stated that this lifestyle is one of several life histories that a pathogen may follow and in many cases human infections represent a dead end. This study focuses on the natural ecology of several human pathogens, V. cholerae, V. parahaemolyticus, and V. metecus, and their associated virulence factors, in regions where they cause sporadic illness as well as a region where one of these pathogens, V. cholerae, has never caused a human illness. In this work we demonstrate the non-human environment as a natural ecosystem for several human pathogens as well as a reservoir of virulence factors. This was achieved by employing a combination of high-throughput whole genome analyses focused on the nucleotide and amino acid level, combined with broader ecological studies evaluating the role of the environment with respect to presence of the pathogens and expression of their virulence factors. This work further demonstrates the ubiquity of virulence factors in the environment and the expression of these factors at temperatures found outside of the human host suggests their utility in the environment

The Effects Of Motility And Chemotaxis On Vibrio Cholerae Colonization In Zebrafish

Vibrio cholerae, the cause of the diarrheal disease cholera, is a gram-negative, curved rod-shaped bacterium, with a single polar flagellum. V. cholerae is naturally found in aquatic environments and is highly motile. When it enters a human host, V. cholerae uses flagellar motility to pass through the stomach and into the small intestine. Once in the small intestine, motility genes are downregulated and virulence gene expression is upregulated. V. cholerae motility and chemotaxis effects have not yet been studied in a zebrafish model, a natural host of this bacterium. We hypothesize that V. cholerae in frame deletions of vital motility and chemotaxis proteins, such as flaA, cheY-3, and motY, would decrease the ability of V. cholerae to colonize the zebrafish intestine. However, the deletion of chemotaxis gene cheY-3 actually significantly increases the ability of V. cholerae to colonize the zebrafish intestine, and only the deletion of motility gene motY significantly decreases colonization compared to wild-type

Water Microbiology. Bacterial Pathogens and Water

Water is essential to life, but many people do not have access to clean and safe drinking water and many die of waterborne bacterial infections. In this review a general characterization of the most important bacterial diseases transmitted through water—cholera, typhoid fever and bacillary dysentery—is presented, focusing on the biology and ecology of the causal agents and on the diseases’ characteristics and their life cycles in the environment. The importance of pathogenic Escherichia coli strains and emerging pathogens in drinking water-transmitted diseases is also briefly discussed. Microbiological water analysis is mainly based on the concept of fecal indicator bacteria. The main bacteria present in human and animal feces (focusing on their behavior in their hosts and in the environment) and the most important fecal indicator bacteria are presented and discussed (focusing on the advantages and limitations of their use as markers). Important sources of bacterial fecal pollution of environmental waters are also briefly indicated. In the last topic it is discussed which indicators of fecal pollution should be used in current drinking water microbiological analysis. It was concluded that safe drinking water for all is one of the major challenges of the 21st century and that microbiological control of drinking water should be the norm everywhere. Routine basic microbiological analysis of drinking water should be carried out by assaying the presence of Escherichia coli by culture methods. Whenever financial resources are available, fecal coliform determinations should be complemented with the quantification of enterococci. More studies are needed in order to check if ammonia is reliable for a preliminary screening for emergency fecal pollution outbreaks. Financial resources should be devoted to a better understanding of the ecology and behavior of human and animal fecal bacteria in environmental waters

<i>Vibrio cholerae</i> non-01 and <i>V. mimicus</i> in diarrhoeal disease : a study of virulence factors

Vibrio cholerae non-01 and V. mimicus, isolated from clinical and environmental sources, were examined for factors related to virulence. The aim was to identify factors which would distinguish pathogenic from non-pathogenic strains and to establish a correlation, if any, with serogroup. The lipopolysaccharide (LPS) of epidemic V. cholerae belonging to serogroups 01 and 0139 are regarded as'virulence factors. In this study certain other serogroups, such as 02,05 and 09 were associated with diarrhoeal disease. LPS of these organisms may have a role in adhesion. Although 90% of V. cholerae non-01 and V. mimicus colonised tissue culture cells, neither smooth LPS, the presence of flagella nor the possession of the toxin coregulated pilus gene (tcpA) were essential for adhesion. Cholera toxin (CT) is the factor responsible for the dramatic symptoms of epidemic cholera and is produced by V. cholerae serogroups 01 and 0139. However, less than 1% of V. cholerae non-01, non-0139 and V. mimicus possessed the gene for CT. Southern blot analysis of the CT genes revealed that most strains carried one CT gene except for serogroups 023 and 0139 which had two hybridising fragments. The variations in fragment size within the 0139 serogroup suggested that this serogroup was not strictly clonal. In addition to CT both V. cholerae non-01 and V. mimicus expressed several other toxins, often concurrently, which made detection of specific toxins difficult. Over 80% of strains produced haemolysin(s) and cytotoxin(s). The cytotoxic effects on tissue culture cells masked cytotonic effects, such as that caused by CT. V. cholerae and V. mimicus did not produce verocytotoxin (VT) and did not possess the genes for VT. The heat-stable enterotoxin (NAG-ST) gene was found in 3% of V. cholerae non-01 and 12% of V. mimicus strains. All V. cholerae strains belonging to serogroup 014, were NAG-ST positive and Southern blot analysis of the NAG-ST genes suggested that this serogroup represented a single clone. In addition to the established toxins two novel factors, which may contribute to the virulence of V. cholerae non-01 and V. mimicus, were found. A novel cytotoxin, produced by 16% of strains, was expressed on Vero cells causing vacuolation of cell cytoplasm. Another putative "toxin" found in 19% of strains was demonstrated in a GMI-ELISA. This "toxin" had the ability to bind ganglioside GMI and shared antibody binding sites with CT. V. cholerae non-01 and V. mimicus also expressed species specific high affinity iron chelating molecules; 74% of V. cholerae non-01 produced enterochelin, whereas 90% of V. mimicus produced aerobactin. Aerobactin production is usually associated with invasive organisms and is unusual among Vibrio spp. None of the virulence factors, except CT, was more prevalent in clinical than in environmental strains. This is perhaps not surprising as vibrio-associated disease is often linked with exposure to the aquatic environment or consumption of seafood. Within V. cholerae the 01 and 0139 serogroups were linked with CT and 014 with NAG-ST, in future, other serogroup - virulence factor associations may be found. For V. mimicus the main virulence factors expressed were aerobactin and NAG-ST. Therefore the pathogenicity of V. cholerae and V. mimicus appears to be multifactorial and it is likely that, as with diarrhoeagenic, E. coli, a heterogeneous pattern of virulence will be found

Vibrio interactions with bivalve hemocytes and analysis of the Crassostrea gigas microbiota

My PhD project aimed at investigating the molecular mechanisms at the basis of the interaction between Vibrio bacteria and shellfish in the bivalve models Crassotrea gigas and Mytilus galloprovincialis and to study the composition and dynamics of bivalve microbiota. Previous studies suggested that persistence of entrapped bacteria inside bivalve tissues depends, at least in part, on their capacity to survive to the hemolymph bactericidal activity, that is exerted by both hemocytes and serum soluble factors. In the first part of my PhD work, hemocytes of M. galloprovincialis were challenged with different pathogenic Vibrio strains (V. aestuarianus 01/032, V. aestuarianus 02/041, V. tasmaniensis LGP32, V. harveyi VH2, V. tapetis CECT 4600 and V. coralliilyticus ATCC BAA 450) in the presence or in the absence of the extrapallial protein present in M. galloprovincialis serum (MgEP), and of the whole hemolymph serum. In addition, C. gigas hemocytes were exposed to the bivalve pathogens V. aestuarianus 01/032 and V. aestuarianus 02/041 under the same conditions to better understand molecular basis of bacteria-hemolymph interactions in oysters. We observed that MgEP promotes D- mannose sensitive adhesion to and killing by hemocytes of the bivalve pathogens V. aestuarianus 01/032, V. aestuarianus 02/041, V. tasmaniensis LGP32 and V. coralliilyticus ATCC BAA 450. In addition, in the presence of M. galloprovincialis EP protein (MgEP), C. gigas haemocytes killed V. aestuarianus 01/032 and V. aestuarianus 02/041 almost as efficiently as mussel phagocytes. These findings suggest that the different sensitivity of Vibrio strains to the antibacterial activity of oyster (susceptible to Vibrio infection) and mussel (resistant to Vibrio infection) haemolymph might partly depend on the fact that C. gigas serum lacks MgEP-like opsonins. These results may have important implications for improving bivalve depuration strategies and prevent diseases affecting bivalve production worldwide. In the second part of my thesis work, I studied the microbial communities associated to contrasting C. gigas samples collected during mortality episodes in different European sites. Real-time PCR targeting oyster pathogens (e.g. Ostreid herpesvirus 1 [OshV-1] and V. aestuarianus) and 16SrRNA gene-based microbial profiling were applied on a large number of C. gigas samples (n=525 and n=101 for qPCR and 16SrRNA gene profiling analysis, respectively) to extensively investigate the patterns and dynamics of oyster microbiota during mortality events. Comparative analysis of contrasting (e.g. infected vs not infected) C. gigas samples conducted using these methods revealed that oyster experiencing mortality outbreaks displayed signs of microbiota disruption associated with the presence of previously undetected potential pathogenic microbial species mostly belonging to genus Vibrio and Arcobacter. This represents to our knowledge, the largest study conducted so far to determine the composition and dynamics of farmed oyster microbiota

Aquatic food security:insights into challenges and solutions from an analysis of interactions between fisheries, aquaculture, food safety, human health, fish and human welfare, economy and environment

Fisheries and aquaculture production, imports, exports and equitability of distribution determine the supply of aquatic food to people. Aquatic food security is achieved when a food supply is sufficient, safe, sustainable, shockproof and sound: sufficient, to meet needs and preferences of people; safe, to provide nutritional benefit while posing minimal health risks; sustainable, to provide food now and for future generations; shock-proof, to provide resilience to shocks in production systems and supply chains; and sound, to meet legal and ethical standards for welfare of animals, people and environment. Here, we present an integrated assessment of these elements of the aquatic food system in the United Kingdom, a system linked to dynamic global networks of producers, processors and markets. Our assessment addresses sufficiency of supply from aquaculture, fisheries and trade; safety of supply given biological, chemical and radiation hazards; social, economic and environmental sustainability of production systems and supply chains; system resilience to social, economic and environmental shocks; welfare of fish, people and environment; and the authenticity of food. Conventionally, these aspects of the food system are not assessed collectively, so information supporting our assessment is widely dispersed. Our assessment reveals trade-offs and challenges in the food system that are easily overlooked in sectoral analyses of fisheries, aquaculture, health, medicine, human and fish welfare, safety and environment. We highlight potential benefits of an integrated, systematic and ongoing process to assess security of the aquatic food system and to predict impacts of social, economic and environmental change on food supply and demand

Mechanisms of virulence associated with thermolabile hemolysin (TLH) from Vibrio alginolyticus on erythrocytes of silver sea bream, Sparus sarba.

Wong, Sze Ki.Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.Includes bibliographical references (leaves 87-106).Abstracts in English and Chinese.Acknowledgements --- p.iAbstract --- p.iiAbstract in Chinese --- p.ivTable of contents --- p.VList of figures --- p.ixList of abbreviations --- p.XChapter Chapter 1. --- General introduction --- p.1Chapter Chapter 2. --- Literature review --- p.6Chapter 2.1. --- Pathogenic mechanisms of Vibrio species in fish --- p.7Chapter 2.1.1. --- Introduction --- p.7Chapter 2.1.2. --- Adhesion --- p.7Chapter 2.1.3. --- Invasion --- p.8Chapter 2.1.4. --- Proliferation --- p.9Chapter 2.2. --- Vibrio virulence factors --- p.12Chapter 2.2.1. --- Introduction --- p.12Chapter 2.2.2. --- Hemolysin --- p.12Chapter 2.2.3. --- Protease --- p.14Chapter 2.2.4. --- Siderophore --- p.15Chapter 2.2.5. --- Lipopolysaccharide --- p.15Chapter 2.3. --- General apoptotic pathways --- p.17Chapter 2.3.1. --- Introduction --- p.17Chapter 2.3.2. --- Extrinsic apoptotic pathway --- p.17Chapter 2.3.2.1. --- Death receptor signaling apoptosis --- p.17Chapter 2.3.2.1.1. --- Fas signaling pathway --- p.18Chapter 2.3.2.1.2. --- TNF-R1 signaling pathway --- p.19Chapter 2.3.2.1.3. --- TRAIL receptors signaling pathway --- p.20Chapter 2.3.2.2. --- Growth factor receptor signaling apoptosis --- p.21Chapter 2.3.3. --- Intrinsic apoptotic pathway --- p.21Chapter 2.3.3.1. --- Mitochondrial apoptotic pathway --- p.21Chapter 2.3.3.1.1. --- Cyto c --- p.22Chapter 2.3.3.1.2. --- Smac/DIABLO --- p.22Chapter 2.3.3.1.3. --- Omi/HtrA2 --- p.22Chapter 2.3.3.1.4. --- AIF and endo G --- p.23Chapter 2.3.3.1.5. --- Bcl-2 family --- p.23Chapter 2.3.3.1.6. --- Mitochondrial membrane permeabilization (MMP) --- p.23Chapter 2.3.3.2. --- p53-regulated apoptotic pathway --- p.24Chapter 2.3.3.3. --- Endoplasmic reticulum (ER) stress-induced apoptotic pathway --- p.25Chapter 2.4. --- Membrane vesiculation in erythrocytes --- p.26Chapter 2.4.1. --- Introduction --- p.26Chapter 2.4.2. --- Induction of vesiculation --- p.26Chapter 2.4.3. --- Contents of vesicles --- p.28Chapter 2.4.4. --- Mechanisms involved during vesiculation --- p.29Chapter 2.4.5. --- Correlation between apoptosis and membrane vesiculation in erythrocytes --- p.31Chapter 2.4.6. --- Reasons for vesiculation --- p.31Chapter Chapter 3. --- "Induction of apoptosis by Vibrio alginolyticus thermolabile hemolysin (TLH) in blood cells of silver sea bream, Sparus sarba" --- p.33Chapter 3.1. --- Abstract --- p.34Chapter 3.2. --- Introduction --- p.34Chapter 3.3. --- Materials and methods --- p.36Chapter 3.3.1. --- Experimental fish --- p.36Chapter 3.3.2. --- Whole blood preparation --- p.37Chapter 3.3.3. --- Preparation of V. alginolyticus TLH --- p.37Chapter 3.3.4. --- "Caspase-3, -8, -9/6 fluorescent assay" --- p.38Chapter 3.3.5. --- TUNEL assay --- p.39Chapter 3.3.6. --- Apoptotic DNA ladder assay --- p.40Chapter 3.3.7. --- Statistical analysis --- p.41Chapter 3.4. --- Results --- p.42Chapter 3.4.1. --- "Increase of caspase-3, -8, -9/6 activities" --- p.42Chapter 3.4.2. --- Detection of DNA fragmentation by TUNEL assay --- p.44Chapter 3.4.3. --- Detection of DNA fragmentation by apoptotic DNA ladder assay --- p.44Chapter 3.5. --- Discussion --- p.46Chapter Chapter 4. --- "Occurrence of membrane vesiculation, apoptosis and post-apoptotic necrosis after exposure to Vibrio alginolyticus thermolabile hemolysin (TLH) in erythrocytes of silver sea bream, Sparus sarba" --- p.51Chapter 4.1. --- Abstract --- p.52Chapter 4.2. --- Introduction --- p.52Chapter 4.3. --- Materials and methods --- p.54Chapter 4.3.1. --- Experimental fish --- p.54Chapter 4.3.2. --- Whole blood preparation --- p.54Chapter 4.3.3. --- Preparation of V. alginolyticus TLH --- p.55Chapter 4.3.4. --- Light microscopy --- p.55Chapter 4.3.5. --- Transmission electron microscopy (TEM) --- p.56Chapter 4.3.6. --- Measurement of membrane vesiculation - acetylcholinesterase (AChE) assay --- p.56Chapter 4.3.7. --- Measurement of necrosis - hemoglobin colorimetric assay --- p.57Chapter 4.3.8. --- Apoptotic DNA ladder assay --- p.58Chapter 4.3.9. --- Flow cytometry --- p.59Chapter 4.3.10. --- Statistical analysis --- p.59Chapter 4.4. --- Results --- p.60Chapter 4.4.1. --- Ultrastructural changes in red blood cells after exposure to TLH --- p.60Chapter 4.4.2. --- Changes of cell population in size and granularity after exposure of TLH --- p.67Chapter 4.4.3. --- Effect of TLH dosage on necrosis and DNA fragmentation --- p.72Chapter 4.4.4. --- "Occurrence of membrane vesiculation, necrosis and DNA fragmentation in cells exposed to TLH" --- p.72Chapter 4.5. --- Discussion --- p.76Chapter Chapter 5. --- General conclusions --- p.82References --- p.8

Exploring Quorum Sensing Dynamics and Biofilm Formation in the Fish Pathogen Aliivibrio salmonicida

The marine pathogen Aliivibrio salmonicida is the causative agent of cold-water vibriosis, affecting mainly farmed salmonid fish when water temperatures are below 10°C. Even though cold-water vibriosis is no longer threatening Norwegian aquaculture, the reemergence of the disease is still a possibility. Therefore, it is crucial to gain knowledge and understanding of the pathogenicity of A. salmonicida. Quorum sensing (QS) is one of the communication systems used by bacteria to regulate gene expression in a synchronized way in response to cell density by secreting and sensing extracellular signals called autoinducers (AIs). QS system controls various physiological processes, particularly virulence system and biofilm formation in many pathogenic bacteria. With the increased emergence of antibiotic-resistant in recent years, understanding and targeting QS system is expected to bring potential new breakthroughs for the prevention and treatment of Vibrio infections. The present work was initiated to increase the knowledge on the QS system and its regulation on phenotypic traits that may be important for survival and host-pathogen interaction in A. salmonicida. Alternative sigma factors such as RpoS provide the main line of responses to changes in the environment by altering gene transcription. In several vibrios, RpoS has been shown to be connected to QS system. The obtained results in this thesis, clearly indicate that an RpoS-like sigma factor, RpoQ (VSAL_II0319) is a component of the QS system and involved in regulating colony rugosity, biofilm formation, and motility in a cell density dependent manner. The transcriptomics analysis further revealed that RpoQ is involved in influencing expression of a large panel of genes including the syp operon involved in polysaccharide production. This suggests that the downregulation of biofilm development and wrinkled colony phenotype were due to RpoQ-dependent repression on polysaccharide biosynthesis genes (syp genes) at high cell density. In addition to cell density dependent control on biofilm formation and colony rugosity through QS, temperature was shown to influence the regulation of RpoQ on these phenotypes, linking this environmental factor to the development of cold-water vibriosis in seawater at low temperatures. Previous reports have shown that A. salmonicida possesses two functional autoinducer synthases, the LuxI and AinS, which are responsible for the production of eight acyl homoserine lactones (AHLs). In this thesis, the inactivation of luxI, but not ainS, led to the formation of wrinkled colonies similar to those formed by the ΔrpoQ mutant. The transcriptome analysis showed that LuxI is required for repression of syp expression, where repression of syp is likely operated through the RpoQ sigma factor. When both systems were inactivated simultaneously, strains (ΔainSluxI−) with wrinkled colonies and mushroom structured biofilm were formed. Furthermore, the exogenous addition of either LuxI, N-3-oxo-hexanoyl-L-homoserine lactone (3OC6-HSL) or AinS, N-3-hydroxy-decanoyl-L-homoserine lactone (3OHC10-HSL), to the ΔainSluxI- double mutant, inhibited biofilm development. This suggested that the downregulation of biofilm formation is operated through a common pathway when the AHL concentrations are high. The results presented in this work, add new knowledge about the nature of the QS mechanism of A. salmonicida and elucidate some aspects of the complex mechanism of biofilm formation, contributing to advancement of research in this field