The Biology of Vibrios

Many Vibrio species are bioluminescent, including Vibrio cholerae and V. fischeri, the genes for bioluminescence having been characterized and detected in Vibrio species by employing gene probes and genomic sequencing. The complete genome sequences of V. cholerae, V.ibrio parahaemolyticus, and V. vulnificus have been determined, providing a rich set of data illuminating the metabolic versatility of these species. Vibrios are clearly very important inhabitants of the riverine, estuarine, and marine aquatic environments. For this reason, by taking the perspective of a global microbial ecology of the vibrios, a deeper understanding of microbial ecological systems can be gained. V. cholerae provides a useful example and is therefore discussed in the context of a general pattern of environmental pathogens and their close linkage with climate, weather systems, seasonality, and physical and chemical parameters. Fully dimensional understanding of an infectious disease, whether cholera, hantavirus, or malaria, reaches from countries to continents and beyond and connects medicine to many viewpoints across science and engineering, and even to daily life. The major cause of death for children 4 years old and under is infectious disease, which causes almost two-thirds, or 63%, of these deaths, and outbreaks of cholera substantially exceed those of any other disease. The current characterization of the ecology of cholera includes global weather patterns, aquatic reservoirs, phages, zooplankton, collective behavior of surface-attached cells, an adaptable genome, and the deep sea, together with the bacterium and its host, emerging from the perspective of biocomplexity.

One or a combination of the bacteriocidal agents is often present in most of the selective media commonly used for the selective isolation of vibrios. A scheme for the isolation of pathogenic vibrios often includes enrichment prior to plating on a selective agar, especially when vibrios are at low levels and competing with other bacteria that can outgrow them in general media. Several enrichment media have been used for the selection of one group or species of Vibrio. Special effort has been devoted to enhance the presence of potential pathogenic species in contaminated food or in clinical samples. Thiosulfate citrate bile salt sucrose agar (TCBS) is an ideal medium for the selective isolation and purification of vibrios. Although this medium was originally designed for the isolation of Vibrio cholerae and Vibrio parahaemolyticus, most vibrios grow to healthy large colonies with many different colonial morphologies. Until now, TCBS was the best selective medium for isolation of vibrios, though some strains of Staphylococcus, Flavobacterium, Pseudoalteromonas, Streptococcus, Aeromonas, and Shewanella may present slight growth on it. TCBS agar is also an excellent medium for the selective isolation of vibrios from environmental sources, but differences in media formulation between manufacturers might be reflected in the enumeration of vibrios. Enumeration molecular techniques include the use of polymerase chain reaction (PCR) analysis or DNA probes for colony hybridization. Often, vibrios recovered directly from cryopreservation require at least two passes to fully recuperate and longer incubation periods.

The taxonomy of vibrios was based on very few morphological features, including flagellation, morphology and curvature of the cells, and cultural aspects. This approach led to the description of many new, poorly characterized species. The DNA-DNA hybridization studies of researchers underpinned the taxonomy of vibrios. This chapter discusses the historical underpinnings of vibrio taxonomy and the traditional phenotypic basis of the taxonomy of Vibrio harveyi group. Subsequently, it provides a review of the recent improvements in the taxonomy of vibrios that are mainly due to the application of genomic methodologies, including amplified fragment length polymorphism (AFLP), repetitive extragenic palindromic polymerase chain reaction (rep-PCR), DNA-DNA hybridization, and multilocus sequence analyses (MLSA). The authors conclude the chapter with a discussion on the development and application of an electronic prokaryotic taxonomy with vibrios as a prototype. The current taxonomy of vibrios is based mainly on genomic data. The taxonomy of vibrios is being rapidly improved owing to the application of modern molecular techniques. New data gathered by these techniques will lead to a revision and improvement of the taxonomy of vibrios in the next few years.

There are numerous reports on typing or differentiation of strains within the same species of Vibrio by various genetic fingerprinting methods. These include ribotyping, restriction fragment length polymorphism, amplified fragment length polymorphism, arbitrarily primed polymerase chain reaction PCR (AP-PCR) or randomly amplified polymorphic DNA PCR, enterobacterial intergenic consensus sequence PCR, pulsed-field gel electrophoresis, and multilocus sequence typing. These techniques require more attention than the gene probe and the PCR techniques and are not suitable for routine examination. Essential genes are conserved in bacteria and other organisms but the sequence may vary, and they are used for phylogenetic analysis. Genetic examination of the Vibrio cholerae strains shows rare strains carrying the toxin genes that are reported to be major virulence genes in other bacterial species. Various 16S rRNA gene-targeted oligonucleotide probes to identify Vibrio vulnificus have been reported. Some strains of Vibrio mimicus share important virulence genes with V. cholerae and less frequently with Vibrio parahaemolyticus. The most frequently used systems are based on the multiplex PCR formats. Practical application of molecular identification techniques is limited mostly to medically important Vibrio species. Selection of suitable target genes and fine-tuning of their detection conditions are the basis for development of more sophisticated molecular identification systems, including multiplex PCR and real-time PCR.

This chapter outlines recent progress in the study of the Vibrio genomes, including information from the genome sequences covered to date. A Japanese group and an American group independently reported the possession of two circular chromosomes for vibrios. First, all the vibrios examined possessed two chromosomes: no vibrios with only one chromosome were found. Second, the size of the large chromosome was relatively constant among the vibrios. The distribution of genes of known function between the large and small chromosomes of vibrios provides tantalizing clues about how the two-chromosome configuration of the Vibrionaceae might confer an evolutionary advantage. Thus, whatever the origin of the small chromosome of vibrios, stable maintenance of genomes with multiple chromosomes might have required the evolution of shared mechanisms to control replication. Genome sequencing of three Vibrio species enabled the authors to precisely compare the genome structures of the strains. The chapter provides a brief introduction to some recent topics on horizontal gene transfer in vibrios, mainly in relation to the acquisition of the genes for pathogenicity. Recently, it was reported that chromosomal superintegrons of vibrios might be a genetic source for the evolution of resistance to clinically relevant antibiotics through integron-mediated recombination events. As with Vibrio parahaemolyticus, further genome sequencing and comparative analysis of more vibrios should give us exciting new knowledge about vibrios exhibiting a variety of lifestyles.

This chapter discusses the collection of gene duplicates (the paranome) in relation to the whole proteome, the functional composition, and organization for all currently available Vibrionaceae genomes, i.e., Vibrio cholerae, V. vulnificus, V. parahaemolyticus, and Photobacterium profundum. The vibrio genomes are organized into two replicons, a main chromosome and an auxiliary chromosome, the latter characterized by less gene synteny than the former. The majority (>77%) of the strain-specific expansion (SSEs) consist of hypothetical proteins or proteins with unknown functions, of which most have no homologous genes outside the Vibrionaceae. The chapter provides an overview of the functional landscape of the paranome for the Vibrionaceae is presented, which allows to determine whether duplicate retention is biased toward specific functional classes for each of the bacterial strains. It appears that the preferentially retained duplicated genes mainly belong to the functional classes that are associated with amino acid metabolism (class E) and transcription (class K). Such genes are directly or indirectly (via regulation) involved in adapting to a constantly changing environment, showing the importance of gene duplicates for biological evolution. Gene duplications can occur on a gene-by-gene basis, resulting in tandem duplicates, or they can result from the duplication of larger regions. Rearrangements after duplication and acquisition of homologs via horizontal gene transfer (HGT) result in duplicates’ being dispersed over the genome.

This chapter discusses the detection of lateral gene transfer (LGT), from the level of individual genes up to whole-genome and multiple locus analyses. The genetic elements most frequently involved in these gene transfers and the genomic hot spots for such events are also described. Lateral DNA transfer is known to occur through three main modes. Many detection methods are used to detect LGT events. The methods for estimating foreign gene content in a genome give different estimates because they detect different laterally acquired gene subsets. The integron/gene cassette system of vibrios is noteworthy, given the substantial contribution it likely makes to LGT. In addition, other integrons with smaller cassette arrays, usually containing antibiotic resistance genes, are frequently found on vibrio plasmids or other genetic elements. Most efforts in studying the evolution of Vibrios have been devoted to pathogenic species. From these studies, many novel mobile genetic elements have been discovered. Several of these elements have been subsequently found in nonpathogenic environmental vibrios, marking them as general tools for vibrio evolution. Much progress has recently been made in trying to quantify laterally acquired DNA in the genomes of vibrios, although many more taxa need to be examined for accurate representation. Vibrio genome sequences and microarray comparisons, phylomes of vibrio genomes, and multilocus sequence analysis of many strains and species of vibrios, allows for a global picture of Vibrio evolution, from the microevolution of conserved housekeeping genes by recombination to mobile genes traveling rapidly between species.

This chapter discusses the specific roles of integrons in the adaptive capacity of the Vibrionaceae, with emphasis on pathogenic Vibrio species and antibiotic resistance. Even though many reports have demonstrated that the presence of antibiotic resistance genes in plasmids or integrons in V. cholerae was the cause of resistance to antimicrobial agents, the mechanism of resistance in other cases was unknown. Integrons likely correspond to one of the most refined tools selected by bacteria, as suggested by the data collected during the last 15 years. The authors recommend using the single term integron to describe all types of integron structures, supporting this suggestion with the fact that the different integrons use the same recombination processes and machinery. The integron gene cassettes for which an activity has been experimentally demonstrated, be they from superintegrons (SI) arrays or from soil DNA, encode proteins related to simple enzymatic functions; their recruitment is seen as providing the bacterial host with an adaptive advantage. Both experimental and phylogenetic data suggest that SIs are the source of the mobile integrons (MI) and resistance gene cassettes observed within clinical isolates.

To possess a functioning flagellar motility system, three things are requisite: a propeller, a motor, and a system of navigation. In this chapter, the swimming and swarming systems are considered separately. There is a central processing system that is shared by both flagellar systems, i.e., chemotaxis, and it allows the bacteria to detect signals in their environment and respond by modifying their movement. Various types of polar flagellar genes and polar flagellar and linked chemotaxis genes have been provided in the chapter. Chemotaxis integrates environmental signaling to modulate behavior by biasing movement toward more favorable conditions or away from unfavorable environments. Chemotaxis integrates environmental signaling to modulate behavior by biasing movement toward more favorable conditions or away from unfavorable environments. One consequence is that not just motility, but also chemotaxis is important for survival and colonization. In Vibrio cholerae, a number of studies show differential expression of various motility and chemotaxis genes under in vivo and in vitro conditions. Bacterial chemotaxis has been most extensively studied in organisms with a few (~6 to 10) peritrichously arranged flagella, e.g., Escherichia coli, Salmonella enterica serovar Typhimurium, and Bacillus subtilis. In swimmer cells, the methyl-accepting chemotaxis proteins (MCPs) localize to both poles; in the swarmer cells, MCPs are found at the poles and at intervals along the cell body. The result is that polar flagellar function influences expression of cell surface polysaccharide, which has important consequences for biofilm formation and host colonization.

This chapter discusses several aspects of adaptation that have been suggested to play a role in the survival of vibrios, in particular, starvation adaptation, the viable but nonculturable (VBNC) response, and biofilm formation. In addition, it talks about quorum sensing, which has been shown to control many phenotypes associated with survival under different conditions. The potential role for oxidative stress in the VBNC response is discussed. Bacteria have evolved complex mechanisms to deal with conditions that are routinely encountered in the natural environment. Such adaptive responses are characterized by changes in gene expression, physiology, and morphology. The potential for nonculturable cells to resuscitate, irrespective of whether the VBNC state is truly a protective strategy or is simply a consequence of stress leading to death, does have implications for the survival of vibrios in the natural environment. A section presents some of the current theories on factors driving the generation and resuscitation of nonculturable cells. The regulation of biofilm formation is complex, involving a range of physical and biological factors, the influences of which vary between species. The chapter provides a review of biofilm formation by Vibrio spp., with a strong emphasis on Vibrio cholerae, for which the most data are available. Biofilm formation on biotic surfaces has implications for the outbreak of disease.

Currently, eight genera are included in the extremophilic Vibrionaceae. According to Bergey’s Manual of Systematic Bacteriology, these genera are Allomonas, Catenococcus, Enhydrobacter, Grimontia, Listonella, Photobacterium, Salinivibrio, and Vibrio. Any luminous spots appearing after overnight incubation generally belong to P. phosphoreum. This organism is an important source of spoilage of modified atmosphere-packed fish products stored at low temperatures, where it respires trimethylamine oxide and produces trimethylamine. One of the challenges in describing low-temperature adaptation in the Vibrionaceae is that many of the model systems that have been used for the studies have undergone taxonomic reclassification. Among the cold shock proteins (CSP), nucleic acid-binding proteins, the CspA family proteins have received the most attention. The isocitrate dehydrogenase (ICDH) isozymes produced by strain ABE-1 (now classified as C. maris) are particularly fascinating examples of psychrophile enzyme adaptation to low temperature. A major breakthrough in studies of the molecular basis of high-pressure adaptation has been the recent report of the complete SS9 genome sequence. V. cholerae preferentially transports glutamate at high osmolarity. Under conditions of external proline, osmotic adaptation in V. vulnificus follows the osmotic induction of proline transport and its conversion to glutamate. In response to starvation conditions at low temperature many bacteria enter into a physiological state in which they cannot be propagated on culture media but remain metabolically active. Such cells have been referred to as having entered the viable but nonculturable (VBNC) state.

This chapter discusses the nature of vibrios--their habitats, ecology, physiological traits, and Evolution. The major components of the isolates identified as gram-negative facultative anaerobes were Colwellia species. Vibrio species are distributed widely and often predominate in the aquatic environment. Rapid die-off occurred after 5 h of incubation, which suggested intraspecific competitions among the microbial populations in the nonsterile sediment environment. This result indicates that the sediment environment is more competitive for Vibrio species to compete with other microorganisms than other habitats in the aquatic environment. Free-living heterotrophic nanoflagellates (HNF) are major and ubiquitous bacterial grazers in various aquatic environments. Vibrio species may use marine animals as their vehicle for survival, where they may escape the grazing pressure of protozoans. The first step of chitin degradation is primarily carried out by microorganisms, and this trait is widespread among many taxonomic groups of prokaryotes. Phenanthrene, a polycyclic aromatic hydrocarbons (PAH), is present in coal tar and petroleum and is a by-product of petroleum refining. Yet it is relevant to note that a marine Vibrio sp. may well be able to degrade PAH. In general, the availability of environmental parameters, such as water temperature, nutrient, and chlorophyll concentrations, is limited, although the use of these data is straightforward in the monitoring of pathogens in aquatic environments. By remote sensing, the sea surface temperature, turbidity, chlorophyll, and sea surface height can be monitored; it has been possible to determine which environmental parameters are strongly linked with epidemics.

Molecular surveys of bacterioplankton communities in coastal regions and open oceans have yielded similar 16S rRNA sequences, although coastal sites can differ significantly from the open ocean with respect to primary production rates and terrestrial influence. While obligate “ultramicrobacteria” have been described from oligotrophic open ocean environments and hypothesized to substantially contribute to environmental nutrient cycling, the extent to which facultative “ultra-micro” Vibrio cells contribute to microbial diversity and nutrient cycling in oligotrophic environments has not been addressed; this may reflect the limitation of DNA-based studies that are based on a collection of planktonic biomass on a 0.2-μm-pore-size filter. Association with larger host organisms may mediate the environmental dynamics of symbiotic or commensal Vibrio populations. Chitinase activity may reflect one of the most important extracellular enzymatic processes in the marine environment. A facultatively anaerobic bacterium originally described as a denitrifying Vibrio was recently classified as an alphaproteobacterium based upon DNA sequence data. Comparative genomic approaches between nonpathogens and pathogenic strains can help explain the unifying themes underlying bacterial-host interactions and mechanisms by which pathogenic interactions may emerge. Environmental genomic approaches to explore the metabolic diversity associated with phylogenetic clades can shed light on how widespread certain features, such as N2 fixation, bioluminescence, and cell signaling, are among the Vibrionaceae and whether vibrios are capable of as-yet- undiscovered metabolic transformations (e.g., denitrification, phototrophy, chemoautotropy). The dynamics and distribution of bacterioplanktonic Vibrio populations are determined by adaptations to environmental gradients, including temperature, salinity, and nutrient concentration.

The light organ symbiosis between the bioluminescent bacterium Vibrio fischeri and the Hawaiian bobtail squid Euprymna scolopes has received increasing interest from researchers representing a range of disciplines, including microbiology, zoology, oceanography, immunology, and genetics. An improved understanding of the factors underlying the natural symbioses can help face problems such as the (re)emergence of diseases with environmental hosts, the effective application of probiotics, overreactions to commensal bacteria by the immune system, and the negative effects of broad-spectrum antibiotics. The light organ is also accessible to experimentally added solutes. Pores connect the outside environment with the site of infection in the light organ, so reagents can be added to cure an infection or monitor biochemical processes at the site of infection. One of the most intriguing colonization factors is bioluminescence. Long considered an energetic drag on cultured cells, luminescence is critical for full colonization of the E. scolopes light organ by V. fischeri. The observation that lipopolysaccharide (LPS) and peptidoglycan monomer (PGM) stimulate developmental processes in a mutualistic animal-bacteria association is notable for at least two reasons. First, the molecules are also recognized by the innate immune systems of animals and often trigger antimicrobial responses. This suggests conserved mechanisms for mutualist and pathogen detection by animals, with host responses being context dependent. Second, the particular PGM molecule shed by V. fischeri is identical to "cytotoxins" of pathogens Bordetella pertussis and Neisseria gonorrhoeae, revealing an interesting and unanticipated similarity between mutualistic and pathogenic bacteria-animal associations.

The abundance and diversity of Vibrio halioticoli and related species in the gut of abalones, and the possible mutual partnership of vibrios in the abalone gut microbial ecosystem, have now been clearly demonstrated. The major habitat of the abalones is the rocky shore with kelp forests >50 m depth. The major food source for the South African abalone Haliotis midae is brown algae, i.e., Ecklonia maxima. It is interesting that most of the nonflagellated vibrios are found in the gut of abalones. Species-specific detection methods are available for V. halioticoli, V. neonatus, and V. ezurae. In situ PCR specific to an alginate lyase gene of V. halioticoli was capable of discriminating between V. halioticoli and related vibrios. The best system for settlement and growth involved use of trail mucus plus diatoms. This led to 97.3% settlement and 70% survival during the 4 weeks of the experiment. Finally, it was apparent that the abalone larvae normally grew up to 1.4 mm in length. Host abalones start ingesting seaweed, preferring brown algae, and this is accompanied by development of the host digestive system. It is reasoned that sustainable nutrient supplies for V. halioticoli and/or other gut microbes lead to stable microbial ecosystem in the gut of abalones. The nonmotile vibrios could then be available for future entry into and colonization of the abalone gut. Volatile short-chained fatty acids (VSCFAs) are available to the host animal as fermentation products converted from energy-rich carbohydrates.

The hypothesis accepted by most coral biologists who study coral bleaching is mass bleaching, which is the result of photobleaching of the endosymbiotic zooxanthellae. Basically, this hypothesis states that the photosynthetic apparatus of the algae is constantly undergoing photodamage in the light. Koch’s postulates were applied to demonstrate that Vibrio shiloi is the causative agent of the bleaching disease of Oculina patagonica. The β-galactoside-containing receptor that V. shiloi recognizes on the O. patagonica surface is present in the coral mucus. The bacteria adhere to a β-galactoside-containing receptor on the coral surface. This was demonstrated by binding the coral mucus to enzyme-linked immunosorbent assay plates: the bacteria adhered avidly to the mucus-coated plates. Electron micrographs of thin sections of O. patagonica following infection with V. shiloi demonstrated large numbers of bacteria in the epidermal layer of the coral. Moreover, using monoclonal antibodies specific to V. shiloi, it was shown that the observed intracellular bacteria were, in fact, V. shiloi. V. coralliilyticus is an etiological agent of bleaching of the coral Pocillopora damicornis on coral reefs in the Indian Ocean and Red Sea. Based on phenotypic and genotypic characteristics, V. coralliilyticus was classified as a new species. Yellow blotch (also called yellow band) disease of the major reef-building coral of the Caribbean Sea, Monastraea spp., is well documented. Coral tumors are not transmitted between colonies, even after fusion of healthy and tumor coral fragments. Studies have indicated that healthy parts of a coral that contains a tumor eventually deteriorate.

The spread of pandemic cholera to South America in the 19th century was probably the consequence of the uncontrolled transport of infected people from regions in Europe where the disease was epidemic. Isolates from the early cholera cases were characterized using a number of phenotypic and molecular typing methods, including multilocus enzyme electrophoresis (MLEE) and ribotyping. These isolates could be distinguished from the seventh pandemic clone by MLEE at just a single locus, leucine aminopeptidase. Epidemic cholera normally occurs in coastal areas and is associated with travelers’ movement worldwide. Several theories were proposed to explain the reemergence of cholera in South America. Two populations of Vibrio cholerae O1 were defined among isolates from 1992 to 1998 in Argentina, based on analysis by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE). Isolates carrying the major virulence genes from the CTX phage and tcpA were linked to the Latin American epidemic clone, whereas isolates lacking these pathogenicity-associated factors and showing distinct RAPD and PFGE profiles characterized the other clone named Tucumán. Growth conditions were optimized for hemolysin production, and the fact that Hcp was also found in this study corroborates the hypothesis of coregulation of both proteins and is evidence of a similar mechanism for the Amazonia strain. Research on cholera and V. cholerae is at a very exciting point. New tools are at hand for a more prompt global genetic analysis of strains, allowing a better description of strains and populations of vibrios.

Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum has been particularly devastating in the marine culture of salmonid fish. The correlation between serotype and virulence may reflect the ability of the bacterial surface antigens to interact with the host tissues. The ferric-anguibactin receptor FatA is an 86-kDa protein that is essential for anguibactin transport. The FatA amino acid sequence is similar to other receptors involved in iron transport, e.g., FhuA and FepA of Escherichia coli, and a TonB box can be identified at its amino-terminal end. V. ordalii causes vibriosis in wild and cultured marine salmonids in the Pacific Northwest of the United States and Japan. The determination and analysis of the complete nucleotide sequence could provide information about the role of pMJ101 in the pathobiology of this fish pathogen. Juvenile salmon exposed to V. ordalii by parenteral challenge developed a systemic infection, and the bacterium was recovered from liver, kidneys, spleen, and blood immediately after the infection. However, the number of bacteria in the liver declined after 1 h and then increased 22 h after infection; bacterial numbers were high in all the organs, and 100% mortality occurred 6 days after infection. The latter observations demonstrate that artificial infection of juvenile salmon is a valid experimental model to study the mechanisms and the bacterial virulence factors involved in the pathogenesis of the infections caused by V. ordalii.

Vibrio harveyi was first described as a species of Acromonobacter by Johnson and Shunk. Later studies in classification of luminous bacteria reported three major groups. The first group contains Photobacterium fischeri, the second group consists of Photobacterium leiognathi and Photobacterium phosphoreum, and the third group contains Beneckea harveyi, Beneckea splendida, and V. cholerae biotype albensis. Luminous vibriosis is the term describing the disease of penaeid prawns caused by luminescent V. harveyi. The use of antibiotics as prophylactic agents to prevent bacterial infection of penaeid larvae has been employed in many shrimp hatcheries. However, the development of antibiotic resistance is one of the major consequences resulting from prophylactic antibiotic use. The use of probiotics is another approach to bacterial control to maintain a beneficial balance of bacteria and other microorganisms in the culture systems. The use of bacteriophage in the control of vibriosis seems very promising. An experiment with bacteriophage from diseased ayu, Plecoglossus altivelis, provided protection against infection by Pseudomonas plecoglossicida, a pathogen of ayu. These bacteriophages, representatives of Myoviridae and Podoviridae, reduced the number of bacterial cells in the kidneys of affected ayu and the underlying water environment. A study conducted by Liu et al. highlighted that V. harveyi was shown to produce proteases, phospholipase, hemolysins, or exotoxins that are important for pathogenicity. Liu and Lee also reported that a cysteine protease is the major exotoxin lethal to the tiger prawn, Penaeus monodon.

Resembling a generalized hemorrhagic septicemia, coldwater vibriosis is characterized by hemorrhaging around the abdomen. Internally, anemia and hemorrhaging of the organs, swim bladder, abdominal wall, and posterior gastrointestinal tract may be seen. Using isolated macrophages from Atlantic salmon and rainbow trout coupled with immunofluorescence techniques, the pathogen was observed to be internalized. Cultures have been recovered from blood and kidney on tryptic soy agar supplemented with 1.5% (wt/vol) NaCl and incubation at 15ºC for up to 5 days. Cultures, which were subsequently named as a new species, Vibrio salmonicida, were recovered from diseased salmon. DNA hybridization of four cultures confirmed homogeneity (DNA homology was 82 to 100%) but with low relatedness to Vibrio anguillarum (30%), Vibrio ordalii (34%), and Vibrio parahaemolyticus (40%). Experimental challenge by means of intraperitoneal injection, immersion, and cohabitation of Atlantic salmon with V. salmonicida has led to the development of clinical disease with pathogenicity related to water temperature. The dihydroxamate siderophore bisucaberin was produced significantly only at ≤10°C, which is when the disease is most troublesome to Atlantic salmon. A proposal was made that temperature-sensitive iron sequestration could well constitute an important virulence mechanism for V. salmonicida. There has been success with the development of whole-cell formalized vaccines, leading to the commercialization of more than one polyvalent product. Immersion of Atlantic salmon in these formulations resulted in protection for at least 6 months, with humoral antibodies being developed to the lipopolysaccharide (LPS) component, particularly the O-side chain.

Bacterial pathogenicity is known to be associated with structural components of the cells or active secretions of substances that either damage host tissues or protect the bacteria against host defenses. Recently, pathologists have begun to describe genes in order to understand the regulation of virulence factors. Nevertheless, little is known about marine bacterial pathogens, and until now there has not been any information concerning the Vibrio splendidus group. V. splendidus was defined initially as a luminous marine species with two biotypes distinguishable by phenotypic features. Genotyping by ribotyping, amplified fragment length polymorphism (AFLP), or PCR-restriction fragment length polymorphism has revealed a remarkably high genetic diversity within this group and suggested its polyphyletic nature. A new concept, the ecotype, has been proposed. This theory is based on the principle of multilocus sequence typing (MLST)-based population genetics. Most V. splendidus-related strains isolated from diseased marine animals and suspected to be the etiological agent of disease have been identified by means of classical phenotypic schemes. The antigenic characteristics of V. splendidus-related strains isolated from diseased turbot or cod have been reported, with authors observing that lipopolysaccharide (LPS) and outer membrane protein profiles were correlated with the origin of strain and the virulence in experimental challenge. Bacterial pathogenicity is associated with structural components of the cells or active secretions of substances that either damage host tissues or protect the bacteria against host defenses.

Vibrios are the scourge of marine and estuarine vertebrates and invertebrates. In some cases the evidence for involvement in disease processes is spurious, whereas other bacterial species are recognized as serious animal pathogens. On the basis of phenotypic and genotypic data, these vibrios were grouped into two new species, Vibrio wodanis and Vibrio viscosus, the latter of which was subsequently reclassified as Moritella viscosa. Pathogenicity has been confirmed in laboratory based challenges involving Chromis punctipinnis. Thus, four to six scales were removed from fish, the dermis was scarified, and the wound was swabbed with 107-108 viable cells of Photobacterium damselae. An immunostimulant, specifically, glucan, enhanced resistance of gilthead bream to experimental challenge with P. damselae subsp. Piscicida. This approach is worthy of further consideration to expand the current range of disease control strategies applicable to pasteurellosis. The authors, Selvin, Lipton and Lee et al, reported that the vibrio was more problematic in shrimp, which were infected with the white spot syndrome virus. A divalent vaccine containing formalized cells and extracellular proteins (ECP) of vibrio alginolyticus was developed. Also, the administration of sodium alginate in the diet improved the resistance of white shrimp (Litopenaeus vannamei) to infection by V. alginolyticus. The newly described species Vibrio coralliilyticus was regarded as pathogenic for coral, Pocillopora damicornis, in the Red Sea. A new bacterial species, Vibrio tapetis, was described to accommodate isolates considered to cause brown ring disease in Manila clams (Tapes philippinarum) and carpet shell clams (R. decussatus).

Cholera is acquired through the consumption of contaminated water or food. After passing through the acid barrier of the stomach, the bacteria penetrate the mucous lining of the small intestine and adhere to intestinal epithelial cells to establish an infection. During colonization of the small intestine, the bacteria begin to express several key virulence factors, including cholera toxin (CT). The action of CT on intestinal epithelial cells is responsible for the massive diarrhea associated with cholera. Key to the ability of Vibrio cholerae to cause disease is the virulence factors that it produces. Extensive research efforts involving innovative techniques have been directed at understanding the mechanisms involved in V. cholerae’s establishing a successful infection. The two most important virulence factors required for the ability of the organism to cause cholera are the CT and the toxin-coregulated pilus (TCP). The evolution of the pathogenic potential of V. cholerae has clearly occurred through the acquisition of several mobile genetic elements. A number of different genetic techniques have been utilized to identify V. cholerae genes important for virulence. Several in vivo transcriptome studies have also shed light on aspects of V. cholerae pathogenesis. Transcriptome profiling of V. cholerae isolated from human stools using microarray analysis has provided the closest understanding of gene expression relevant to the natural host. Considering the severe global impact of cholera, much effort has been directed at vaccine development.

Vibrio parahaemolyticus, a gram-negative marine bacterium, causes seafood-borne gastroenteritis in humans. The major source of V. parahaemolyticus infection is contaminated seafood or seafood products. The whole genome sequence of the clinical V. parahaemolyticus strain RIMD2210633 was recently reported. The most notable finding in relation to the pathogenicity of V. parahaemolyticus was the presence of two sets of genes for the type III secretion system (TTSS) in the genome. TTSS is an apparatus of gramnegative pathogenic bacteria used to secrete and translocate virulence factor proteins into the cytosol of target eukaryotic cells. To determine whether the two sets of the TTSS genes identified in the genome of V. parahaemolyticus are functional, a series of mutant strains from strain POR-1 was constructed. POR-1 was used as the parent strain for further mutant construction to exclude the influence of thermostable direct hemolysin (TDH) production on bacterial phenotypes. To examine the role of TTSS1 and TTSS2, specific genes were disrupted using inframe deletions by homologous recombination with the positive selection suicide vector pYAK1. The genes thus disrupted were vcrD1 and vcrD2, each encoding an inner membrane protein; vscC1 and vscC2, each encoding an outer membrane protein; and vscN1 and vscN2, each encoding a cytoplasmic protein, of the TTSS apparatus. Genome sequencing of V. parahaemolyticus and subsequent studies have shed light on hitherto unknown aspects of the pathogenicity of this organism. Both of the two sets of TTSS in KP-positive V. parahaemolyticus are functional and are involved in distinct phenotypes.

This chapter discuss the taxonomy, infections, pathogenesis, genetic heterogeneity, distribution in estuarine environments and the environmental parameters that contribute to the ecology of this organism, and methods to eliminate this pathogen from foods. Of the several human pathogens now realized to occur naturally in seawater, the most significant, in regard to virulence, is Vibrio vulnificus. Three biotypes of V. vulnificus are recognized. Whereas each is known to be a human pathogen, biotype 1 is almost exclusively associated with human disease, and this is the biotype of greatest public health concern. Since the first study on experimental pathogenesis in V. vulnificus, a considerable amount has been learned regarding the virulence factors important for V. vulnificus infection. The major symptoms associated with V. vulnificus infections, including fever, tissue edema, hemorrhage, and especially hypotension, are classic symptoms associated with endotoxic shock. A study that examined 62 biotype 3 strains from Israel, as well as 82 biotype 1 and 15 biotype 2 strains, indicated that biotypes 1 and 2 are not entirely distinct but are present in two genetic subpopulations. While thiosulfate citrate bile salts sucrose agar (TCBS) is the most commonly employed medium for the isolation and initial differentiation of marine vibrios, most studies on the distribution of V. vulnificus in marine environments now employ colistin-polymyxin B-cellobiose agar or one of its modifications.

Besides three clinically important species in the genus Vibrio-V. cholerae, V. parahaemolyticus, and V. vulnificus-nine other Vibrio species have been isolated from human infections. They include V. mimicus, Grimontia hollisae, V. fluvialis, and V. furnissii, which are solely or principally isolated from gastroenteritis cases, and V. alginolyticus and Photobacterium damselae, which are chiefly associated with wound infections. In addition, V. metschnikovii, V. cincinnatiensis, and V. harveyi are chiefly isolated from extraintestinal infections, but these isolations are less frequent. The nine species are currently considered to be potential human pathogens and are reportable in public health surveys from the marine environment and seafood. The rare isolation of Grimontia species is partly due to its unique growth and biochemical characteristics. G. hollisae does not grow, or grows very poorly, on selective isolation media for enteric pathogens, including thiosulfate citrate bile salt sucrose (TCBS) agar and MacConkey agar. Two factors in G. hollisae that may play roles in diarrhea have been studied. G. hollisae produces a hemolysin similar to the TDH (thermostable direct hemolysin) of V. parahaemolyticus. Extracellular proteins (ECPs) and cell-associated factors have been studied in experimental models to determine if V. furnissii produces a possible diarrheagenic factor(s), with results indicating that culture filtrates of some strains cause diarrhea and mortality in suckling mice. The virulence of other cultures may depend on the production of other virulence factors, the nature of which differs from strain to strain.

This chapter summarizes available information on the epidemiology of major clinically significant Vibrio species, including Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus with an especial emphasis on cholera. Hallmarks of the epidemiology of cholera include a high degree of clustering of cases by location and season, with highest rates of infection in children 1 to 5 years of age in endemic areas, and protection against the disease being afforded by improved sanitation/hygiene and preexisting immunity. V. cholerae strains associated with epidemics also undergo frequent genetic and phenotypic changes. Surveillance of cholera in Bangladesh has provided important information regarding the epidemiology and seasonality of the disease. Developments in DNA analysis techniques led to the introduction of several new typing methods that have enabled the study of the epidemiology of V. cholerae on a global scale. Subsequently, it was proposed that, in addition to other possible seasonal factors causing a bloom of diverse V. cholerae in the environment, epidemics may be preceded by a gradual enrichment of pathogenic strains through passage in human beings who consume surface water. Diarrheal disease due to V. parahaemolyticus is toxin-mediated. At least two toxins have been identified as potential virulence factors, thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH). Preventive measures to interrupt transmission of pathogenic vibrios and thus reduce the disease burden are possibly the most effective disruption to the emergence or rapid evolution of Vibrio species toward enhanced virulence.

This chapter provides a summary of the main applications which have been commercialized or which are being developed as biotechnological products or processes. Two oral vaccines are available. First is the WC/rBS vaccine, which consists of whole cells of Vibrio cholerae O1 plus a recombinant subunit of the cholera toxin. This provides good protection. Second, a live attenuated vaccine is available which is a genetically manipulated V. cholerae strain, CVD103-HgR. As well as being useful for the development of cholera vaccines, there are also some interesting applications being looked at which use Vibrio-derived toxins in other ways. The discovery of quorum sensing in species of Vibrio in the 1980s is one of the most important developments in the history of microbiology. The evolution of quorum sensing was a major step which allowed bacteria to secure some of the adaptive advantages of more complex multicelled organisms. Vibrio spp. have been found to produce a variety of extracellular proteases. V. alginolyticus, for example, produces six proteases, including an unusual detergent- resistant alkaline serine exoprotease. However, an enzyme-linked immunosorbent assay for the detection of V. cholerae in a variety of homogenized samples has been developed.

The limitation of agar plates to recover microorganisms from environmental samples was realized over a century ago by Winogradsky. Indeed, the great majority of the marine microbiota is elusive to growth on conventional laboratory media, probably due to the artificially high nutrient load of commercial media and incompatibility of the media with the environmental conditions. It is clear that there is still a widespread reliance on thiosulfate citrate bile salt sucrose (TCBS) agar for the recovery of vibrios, despite its formulation being specifically for cholera. Recent studies have shown that quantitative data obtained using TCBS parallel the molecular counts by quantitative PCR. Biofilm formation is an important feature in the ecology (and pathogenesis) of vibrios. The development of biofilms is under orchestrated and complex genetic control involving several independent loci. Phenotyping techniques will probably give place to molecular tools for the needed screening of massive numbers of isolates. Multilocus sequence analysis (MLSA) and whole-genome sequence data will also allow vibrio taxonomists to address questions concerning the species definition and concept, a highly debatable issue in the current prokaryotic taxonomy. The usefulness of genome-based approaches to taxonomy, using amino acid identity of MLSA data, may offer insights on vibrio systematics. Lipopolysaccharide and peptidoglycan monomer (PGM) molecules normally trigger antimicrobial responses by the innate immune system of animals, but in the specific case of V. fischeri and E. scolopes, the host seems to tolerate the benign infection.