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Biodiversity of Vibrios

Laboratory of Microbiology and,¹ BCCM/LMG Bacteria Collection Ghent University, Ghent, Belgium,² Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan³

SUMMARY

INTRODUCTION: HISTORICAL ASPECTS

OCCURRENCE AND IMPORTANCE

Human Pathogens

Coral Pathogens

Nutrient Cycling

Role in Aquaculture

ISOLATION AND MAINTENANCE

GENOTYPIC IDENTIFICATION

Amplified Fragment Length Polymorphism

Colony Hybridization by Species-Specific Probes

Fluorescence In Situ Hybridization

Microarrays

Multilocus Enzyme Electrophoresis and Multilocus Sequence Typing

Random Amplified Polymorphic DNA and Repetitive Extragenic Palindrome PCR

Real-Time PCR

Restriction Fragment Length Polymorphism

Ribotyping

PHENOTYPIC IDENTIFICATION: THE PITFALLS OF CLASSICAL BIOCHEMICAL IDENTIFICATION AND DICHOTOMOUS KEYS

NUMERICAL AND POLYPHASIC TAXONOMY

PHYLOGENY OF THE VIBRIOS

Application of 5S rRNA as a First Phylogenetic Attempt

Phylogenetic Picture Obtained by the 16S rRNA Chronometer

New Phylogenetic Insights Obtained by Other Chronometers

DEFINING TAXA WITHIN THE VIBRIOS

The Families Vibrionaceae, Enterovibrionaceae, Photobacteriaceae, and Salinivibrionaceae

Genera within the Vibrionaceae

Species within the Vibrionaceae

GENOMIC DIVERSITY AND PHYLOGENY AS REVEALED BY IDENTIFICATION METHODS: TOWARD A SYNTHESIS

PLASTICITY OF VIBRIO GENOMES

Genome Configuration

Driving Forces in the Evolution of Vibrios

PERSPECTIVES AND EXPLOITATION

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION: HISTORICAL ASPECTS

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In 1854, the Italian physician Filippo Pacini (1812 to 1883) discovered the first Vibrio species i.e., V. cholerae, the causative agent of cholera, while studying outbreaks of this disease in Florence. Records of a cholera-like disease may well be traced back to the times of Hippocrates (460 to 377 BC) (38). Pacini examined the intestinal mucosa of fatal victims of cholera by using a microscope and detected V. cholerae in all samples. He further pointed out that cholera was a contagious disease, but at that time most scientists and physicians believed in the miasmatic theory of disease (the theory of disease causation by bad or pestilential airs). In the same period, John Snow (1813 to 1858) studied the epidemiology of cholera in several cities of England including Birmingham, London, and Manchester (359; available online at http://www.ph.ucla.edu/epi/snow/snowbook.html). Cholera had killed tens of thousands of people in England between the 1830s and 1850s. According to Snow, cholera was propagated by a "morbid poison entering the alimentary canal." The poison was in (polluted) drinking water. He recommended the provision of pure tap water, free of contamination by sewers and house drains, as an effective means of containing the dissemination of the disease.

Nearly 30 years latter, Robert Koch (1843 to 1910) obtained pure cultures of the deadly V. cholerae on gelatin plates. In August 1883, Koch and his team went to Egypt, where cholera had broken out and caused about 100,000 casualties. In Alexandria, they examined a number of fatal cases and always found a characteristic bacterium in the tissue of the intestine, but they were not able to grow the organism. Subsequently, Koch and his team went to India, and by the end of 1883 they had obtained pure cultures of V. cholerae. They also described some properties of the organism: "It is a little bent resembling a comma or a spiral. It is highly motile and swarms on gelatine plates" and concluded that this organism was indeed the causative agent of cholera (47). In 1893, an outbreak of cholera occurred in Hamburg, Germany, with about 8,000 fatal cases. Koch was requested to study means of providing improved hygiene in that region. He proposed that water supply systems should incorporate filtration of drinking water in order to remove the bacteria. At that time, Koch and his team also realised that vibrios were ubiquitous in aquatic settings and that many "forms" of vibrios were non-pathogenic for humans (47). The first nonpathogenic Vibrio species, i.e., V. fischeri, V. splendidus, and Photobacterium phosphoreum isolated from the aquatic environment, were discovered by the Dutch microbiologist Martinus Beijerinck (1851 to 1931) in the late 1880s.

OCCURRENCE AND IMPORTANCE

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According to Bergey's Manual of Determinative Bacteriology (1994) and Bergey's Manual of Systematic Bacteriology (in press), vibrios (Vibrionaceae strains) belong to the Gammaproteobacteria, are gram negative, usually motile rods, are mesophilic and chemoorganotrophic, have a facultative fermentative metabolism, and are found in aquatic habitats and in association with eukaryotes. They are generally able to grow on marine agar and on the selective medium thiosulfate-citrate-bile salt-sucrose agar (TCBS) and are mostly oxidase positive.

Vibrios are highly abundant in aquatic environments, including estuaries, marine coastal waters and sediments, and aquaculture settings worldwide (20, 95, 164, 297, 298, 321, 399, 446). Several cultivation-dependent and independent studies have showed that vibrios appear at particularly high densities in and/or on marine organisms, e.g., corals (331), fish (6, 146, 179, 325), molluscs (345), seagrass, sponges, shrimp (133, 404-406) and zooplankton (163, 194, 365, 408, 409).

Photobacterium leiognathi and P. phosphoreum are found in symbiotic associations with fish, and P. leiognathi, V. logei, and V. fischeri are found in symbiotic associations with squid. These bacteria colonize the light organs of the host and play a role (via emission of light) in communication, prey attraction, and predator avoidance (120, 126, 339). In the light organs of the squid Sepiolla spp., the abundance of vibrios can be as high as 10¹¹ cells/organ (120, 285). Newly hatched squid excrete a mucus matrix from the pores of the light organs whereby V. fischeri cells present in sea water are caught (94, 289, 290). Subsequently, V. fischeri migrates into the organ and colonizes the crypt epithelium. Obviously, the flagella of V. fischeri play a crucial role in the colonization of the light organs, but hyperflagellated V. fischeri cells containing up to 16 flagella are defective in normal colonization (265). V. fischeri, V. logei, and P. leiognathi are apparently the only three organisms colonizing the light organs of squid, but this seemingly specific partnership remains to be confirmed. V. fischeri cells entrapped in the light organs of squid can sense the density of conspecific cells by signaling molecules or pheromones (e.g., N-acyl homoserine lactones) and thereby regulate bioluminescence (436). This cell-to-cell communication, or "quorum sensing," may play a role in the evolution of symbiotic bacteria (373). It has also been documented for the pathogens V. anguillarum (268), V. cholerae (53, 155, 451), V. harveyi (237, 252), V. parahaemolyticus (165), and V. vulnificus (259). These bacteria monitor cell density and regulate the expression of virulence genes by means of quorum sensing. Luminescence and virulence seem to be coregulated in V. harveyi, and therefore the infections caused by this organism in shrimp could be controlled by signaling antagonists produced by the alga Delisea pulchra (252).

Large numbers of Vibrio and Photobacterium (4.3 x 10⁶/mm²) attached to the external surface of zooplankton have also been reported (163). It has been suggested that a close partnership occurs between these bacteria and zooplankton. The biofilm formation by Vibrio spp. on the exoskeletons of these crustaceans and other marine organisms may in fact constitute a strategy to survive during starvation and/or other environmental stresses (238, 416). In biofilms these bacteria can use trapped and absorbed nutrients, resist antibiotics, and establish favorable partnerships with other bacteria or hosts. Copepods may, in turn, feed on these bacteria. V. cholerae moves along and attaches to surfaces with the aid of the flagellum and pili, which may act as adhesins. In as little as 15 min, V. cholerae forms microcolonies on surfaces; subsequently it produces exopolysacharides, which stabilize the pillars of the biofilm. A 15-µm-thick biofilm, with pillars of cells and water channels, is formed within 72 h (273, 423-425). According to these authors, the strong ability of V. cholerae E1 Tor to form densely packed biofilms in the environment gives a survival advantage to this organism, which is the predominant cause of cholera. Because V. cholerae is closely associated with plankton, it is assumed that cholera outbreaks are linked with planktonic blooms and the sea surface temperature, and so such outbreaks may be predicted by monitoring these parameters by e.g., remote sensing (238). The wide ecological relationships and ability to cope with global climate changes may be a reflection of the high genome plasticity of vibrios (238). Recently, a number of reports have highlighted the pathogenic potential of vibrios toward humans and marine animals (e.g. corals, gorgonians, and shrimp), which may be coupled with rising of sea water temperature due to global warming (215, 253, 331, 350).

V. parahaemolyticus causes gastroenteritis in which the hemolysins, thermostable direct hemolysin (TDH) and/or TDH-related hemolysin (TRH), have been considered to play a crucial role (171, 195, 284, 371). It has been suggested that V. parahaemolyticus acquired the genes encoding these hemolysins via horizontal gene transfer (284, 303). Raimondi et al. (317) have proposed that TDH acts as a porin in the enterocyte's plasma membrane and allows the influx of multiple ionic species, e.g., Ca²⁺, Na⁺, and Mn²⁺. A high concentration of TDH increases the number of porin channels, and this, in turn, results in ionic influx, culminating in cell swelling and death due to osmotic imbalance (317).

Whole-genome sequencing of V. parahaemolyticus (251) revealed that the organism possesses two sets of genes for the type III secretion system (TTSS). TTSS is one of the bacterial protein secretion systems that secretes bacterial proteins into the extracellular environment, but it can also inject bacterial proteins directly into target host eukaryotic cells (175). TTSS is essential for the pathogenicity of bacterial pathogens such as Salmonella, Shigella, Yersinia, and plant pathogens, which cause disease by intimate interactions with eukaryotic cells. However, TTSS is absent from the genome of V. cholerae (162). Gene disruption experiments demonstrated that the two TTSSs of V. parahaemolyticus are functional and that they play a role in the pathogenicity of the bacterium (K. S. Park, T. Ono, M. Rokuda, M. H. Jang, K. Okada, T. Iida, and T. Honda, submitted for publication). Other toxins, proteases, cytolysins, and pili may also play a role as virulence factors in both V. parahaemolyticus and V. vulnificus. In the genome of V. parahaemolyticus (251), other genes that may be involved in pathogenicity have been identified. These genes include those used for bacterial adherence and biofilm formation, such as the genes for the biosynthesis of several pili and the tad genes (198) and for several toxin homologues including an RTX toxin. Certain strains of V. parahaemolyticus, probably derived from a common clonal ancestor, have recently caused a pandemic of gastroenteritis (58, 59, 69, 91).

V. vulnificus is an important etiologic agent of wound infections and septicemia in humans (59a, 122). This sort of septicemia occurs mainly in immunosuppressed people and in patients with high levels of serum iron (caused by genetic mutation, e.g., hemochromatosis, or by liver diseases, e.g., cirrhosis). Iron seems to enhance the virulence of vibrios. A capsular polysaccharide (CPS) is the primary virulence factor in V. vulnificus pathogenesis (270, 422, 441). The presence of this factor correlates with the opaque colony phenotype and is thought to play a inflammatory role within the human body. Smith and Siebeling (357) described four essential genes, i.e., wcvA, wcvF, wcvI, and orf4, responsible for the synthesis of CPS. They showed that mutation in any of these genes results in loss of capsule, which is typical of an avirulent translucent colony phenotype (441). Two lytic bacteriophages, i.e., CK-2 and 153A-5, have been successfully used to treat local and systemic infections caused by V. vulnificus in mice (64). A dose of 10⁸ phage/mice significantly reduced the number of V. vulnificus organisms isolated from wounds and liver of mice. Estrogen seems to provide protection against V. vulnificus lipopolysaccharide-induced endotoxic shock in rats, halving the mortality rate of infected animals (263).

Other vibrios, e.g., Grimontia hollisae, P. damselae, V. alginolyticus, V. cincinnatiensis, V. fluvialis, V. furnisii, V. harveyi, V. metschnikovii, and V. mimicus, have been sporadically found in human infections (1, 46, 56, 89, 113, 114, 443). Apparently, they are less important as human pathogens (113, 114).

Coral reefs are important sources of income for several countries via tourism and fishing, but they also represent protection to coastal areas and may be a source of biologically active compounds e.g., antimicrobials and antivirals. Tourism in the Caribbean generates nearly 90 billion dollars annually (169). Corals reefs may be used as clarifying agents in the sugar industry and as as building materials (228). Some 30 coral diseases (3 of them caused by microbial consortia) have been documented so far, but only 5 have fulfilled the Koch's postulates (370). Coral bleaching, i.e., the paling or the loss of color due to the disruption of symbiosis between the coral host and symbiotic Zooxanthellae, is one of the most serious diseases affecting corals worldwide (332, 370), although it is sometimes reversible in 3 to 6 months (48, 83, 308, 322, 323, 370).

Coral bleaching is thought to be linked to the increased seawater temperature due to recent global climate changes caused by greenhouse gas emissions, although other factors such as seawater eutrophication by sewage and aquaculture, sedimentation, light (UV radiation and photosynthetically active radiation), pollution by heavy metals, and reduction of salinity may also play a role (332). The strongest bleaching episodes have occurred during El Niño years, when surface seawater temperatures reach maxima higher than the summer maximum. The pivotal role of bacteria in coral bleaching and the effect of temperature in bacterial virulence have been studied by Rosenberg and collaborators (32, 33, 331, 333). V. shilonii (also known as V. mediterranei) and V. coralliilyticus have been proven to bleach corals, and their pathogenicity was shown to be temperature dependent.

V. shilonii was identified as an etiological agent of the bleaching of Oculina patagonica, and the main disease steps, i.e., adhesion, penetration, and multiplication (up to 10⁹ CFU/cm³ in 5 days) within the coral tissues have been described in detail (18, 19, 331). Within the coral tissues, most V. shilonii cells become viable but nonculturable (VBNC) but continue to be virulent. According to Sussman et al. (369), the fire worm Hermodice carunculata is a winter reservoir and summer vector of V. shilonii.

V. coralliilyticus, another temperature-dependent pathogen, was shown to cause patchy necrosis of tissues of Pocillopora damicornis when the coral was incubated at temperatures of 27°C or higher (33). Because tropical seawater temperatures have undergone warming in the past 100 years and increases of 1 to 2°C have been predicted by 2100 as a result of increased emission of greenhouse gases, it is expected that bleaching episodes will occur even more frequently (169). Infectious diseases may reduce the global diversity of corals (241).

Recent work on the diversity of vibrios associated with coral bleaching in Davies Reef and Magnetic Island (Great Barrier Reef, Australia) and in the Kaneohe Bay (Hawaii) indicated that different species, i.e., Enterovibrio coralii, P. eurosenbergii, V. fortis, V. campbellii, V. harveyi, V. mediterranei, and V. rotiferianus, may be involved in the process of coral bleaching (F. L. Thompson, D. Gevers, P. Dawyndt, C. C. Thompson, S. Naser, B. Hoste, and J. Swings, submitted for publication; F. L. Thompson, C. C. Thompson, S. Naser, B. Hoste, C. Munn, D. Bourne, and J. Swings, submitted for publication). V. harveyi has been implicated in the disease of a wide range of marine animals, including bleaching in O. patagonica and white band disease in Acropora cervicornis (14, 139, 370).

The mode of infection in fish consists of three basic steps (14, 223, 224): (i) the bacterium penetrates the host tissues by means of chemotactic motility; (ii) within the host tissues the bacterium deploys iron-sequestering systems, e.g., siderophores, to "steal" iron from the host; and (iii) the bacterium eventually damages the fish by means of extracellular products, e.g., hemolysins and proteases. Grisez et al. (145) showed that infection of turbot (Scophthalmus maximus) larvae by V. anguillarum occurs in the intestinal epithelium, where the pathogen invades the bloodstream and spreads to different organs, culminating in death of the fish. More recently, Ringo et al. (326) detected bacterial endocytosis in the pyloric ceca and midgut of arctic charr (Salvelinus alpinus L.) adults and suggested that the whole gastrointestinal tract of fish may be subject to infection.

Internal symptoms of disease in fish caused by strains of vibrios include intestinal necrosis, anemia, ascitic fluid, petechial hemorrhages in the muscle wall, liquid in the air bladder, hemorrhaging and/or bloody exudate in the peritoneum, swollen intestine, hemorrhaging in or on internal organs, and pale mottled liver (14). External symptoms include sluggish behavior, twirling, spiral or erratic movement, lethargy, darkened pigment, eye damage/exophthalmia, hemorrhaging in the mouth, gill damage, white and/or dark nodules on the gills and/or skin, fin rot, hemorrhaging at the base of the fins, distended abdomen, hemorrhaging on the surfaces and muscles, ulcers, and hemorrhaging around the vent.

Using a very robust crustacean model organism, i.e., Artemia spp., and with the aid of transmission electron microscopy, Verschuere et al. (410) established the infection route of V. proteolyticus CW8T2. These investigators first infected Artemia nauplii by inoculating the pathogen in the rearing water. One day later, they detected bacteria that had penetrated "in" the gut epithelium, with subsequent tissue damage, qualified by the authors as "devastating," and had spread toward the host's body cavity. This study illustrates well the infectious capability of certain Vibrio strains and suggests that vibriosis in penaeid shrimp larva rearing systems would be even more devastating, taking into account the fragility of these larvae. Lavilla-Pitogo et al. (225) have reported massive losses in shrimp cultures in Philippines due to a so-called group of "luminous vibrios." According to these authors, there was a decrease of nearly 60% in the survival of reared shrimp between 1992 and 1994, associated with the presence of luminous vibrios in rearing water. Lavilla-Pitogo et al. (225) recommended to farmers that shrimp rearing should not start unless luminous vibrios were absent. The rationale that all luminous vibrios are invariably associated with disease outbreaks in shrimp rearing contrasts with the results obtained by Fidopiastis et al. (120, 121), McFall-Ngai (260, 261), Oxley et al. (301), and Ruby (339), among others, who have reported beneficial and/or harmless partnership between certain luminous vibrios e.g. V. logei and V. fischeri and host invertebrates. For instance, Oxley et al. (301) examined the bacterial flora of healthy wild and reared Penaeus mergulensis shrimp and found a high abundance of vibrios (including V. logei at ca. 10⁴ to 10⁵ CFU/g of gut). The authors also found that the bacterial floras of wild and reared penaeid shrimp are similar and suggested that shrimp may influence and/or select the composition of their gut microbiota. In the light of the current knowledge about the bacterial population structure of certain human pathogens, e.g., Neisseria spp. (256), it is more likely that under favorable conditions (e.g., high nutrient loads and high animal density) within rearing systems, a certain hypervirulent strain (or clone) dominates the bacterial community and causes disease in fish and shellfish rather than the disease being caused by the whole bacterial species. This view implies that only a minority of Vibrio strains are true pathogens and further underscores the idea that many Vibrio species are opportunistic pathogens.

The pathogenic effects of certain strains of vibrios are critical in aquaculture settings, where organisms, e.g., penaeid shrimps and salmonids, are reared at high densities under very artificial and unstable conditions (14, 35, 295). To maintain the productivity of such an intensive aquaculture, high inputs of fish protein originating from the sea have to be employed for feeding, together with high levels of water exchange and the massive use of antibiotics. It seems that the combination of these conditions favors the proliferation of vibrios and enhances their virulence and disease prevalence. This highly intensive aquaculture has disastrous effects for the environment (132, 279, 280, 431). According to Nailor et al. (279, 280) some of the most serious negative environmental impacts are (i) loss of wild fish (5 kg of wild fish has to be caught to feed 1 kg of carnivorous fish reared), (ii) loss of natural habitats (e.g., mangroves), (iii) use of wild seed to stock ponds, (iv) impact of foreign fish and shellfish introduced in the wild, and (v) effluent discharge and coral reef degradation. The spread of antibiotic resistance from aquaculture settings to natural environments has recently been shown (154, 178, 235, 419). About 70% (n = 100) of the vibrios isolated from aquaculture settings in Mexico are multiple-drug resistant (271, 330). Several Vibrio isolates have acquired resistance to the most commonly employed antibiotics (e.g., enrofloxacin, florfenicol, trimethoprim, and oxytetracycline) in shrimp rearing, suggesting that the recently initiated application of these antimicrobials has led to the generation of resistant strains of vibrios (271, 330). Ben-Haim et al. (34) have advanced the hypothesis that aquaculture settings serve as foci or reservoirs for pathogenic Vibrio strains: during certain periods of the year, pathogenic vibrios would endure adverse environmental conditions within aquaculture settings and when favorable environmental conditions are reestablished, vibrios would be able to cause disease in wild animals.

Alternatives involving more environmentally sound aquaculture have been proposed (35). Vaccination has been successfully used to control V. anguillarum and V. vulnificus infections in fish (49, 124). Because certain Vibrio strains may be potential probiotics and/or symbionts of commercially important organisms such as penacid shrimp, salmonids, flatfish, oysters, and abalones, recent studies have suggested that such strains could act as biocontrol agents in aquaculture, diminishing the need for antibiotics and reducing effluent discharges (99, 327, 411). The normal bacterial community associated with L. vannamei has recently been examined in order to find potential probiotic organisms (133-135, 274, 404, 405). Planktonic and particle-associated vibrios seem to enhance the survival and growth of reared L. vannamei. Moss et al. (274) reported that Vibrio and Aeromonas compose up to 85% of the bacterial flora in the gut of this shrimp (about 10⁹ CFU/g of gut tissue), whereas Gomez-Gil et al. (133) found a wealth of vibrios, i.e., 10⁵ CFU/g and 10⁴ CFU/ml, respectively, in the hepatopancreas and hemolymph of healthy L. vannamei.

Pujalte et al. (313) have reported a dominance of vibrios associated with cultured oysters: up to 6.5 x 10⁵ CFU/g of oyster but only 10² CFU/ml in rearing seawater. Using fluorescence in situ hybridization (FISH), the same authors determined that vibrios accounted for up to 40% (156 cells/ml) of the heterotrophic culturable flora grown on marine agar. In a successful recirculating rearing system for rotifers, the abundance of Vibrio spp. was up to 1.7 x 10⁵ CFU/ml, suggesting that these bacteria were playing a positive role in the health of the rotifers (365). These strains were later classified as a new species, V. rotiferianus (136).

Sawabe et al. (345) estimated the abundance of V. halioticoli strains in the gut of several abalone (Haliotis) species. They reportd that V. halioticoli is the dominant culturable bacterium, representing 40 to 64% of the total heterotrophic community, which varied from 10³ to 10⁷ CFU/g of gut. V. halioticoli strains were found to produce large amounts of acetic and formic acids (up to 68.1 mM), which may in turn be used as an energy source or precursor for protein synthesis by the abalones. The authors suggested that a mutual relationship may exist between V. halioticoli and abalones (173).

Because the use of probiotics for humans and domestic animals, e.g., pigs and chickens, has had a certain success (377), several researchers advocate that the use of probiotic bacterial strains or selected mixtures will have a positive impact on health management in marine organisms (10, 295, 411). A considerable difference between the culture of domestic and aquatic animals is that the latter are in constant and intimate contact with a wealth of microrganisms, e.g., viruses, protozoa, and fungi (352, 353). Unfortunately, studies of the use of "probiotic" bacteria have not looked at the interactions with the aquatic microbial food web (16, 352). The so-called probiotic bacterial strains could well be fueling the food web, giving rise to a high abundance of e.g., protozoan flagellates and ciliates, which in turn would be grazed by fish and/or shellfish larvae, improving their survival and growth (383).

ISOLATION AND MAINTENANCE

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Vibrios are fairly easy to isolate from both clinical and environmental material, although some species may require growth factors and/or vitamins. There are several commercial media which may be used for the isolation of vibrios, but tryptone soy agar (Oxoid or Difco) supplemented with 1 to 2% NaCl and marine agar (Difco) generally allow the growth of very healthy colonies after 1 to 2 days of incubation at 28°C. Vibrios grow well at temperatures between 15 and 30°C, depending on the strain under analysis. Obviously, psychrophilic vibrios, i.e., V. logei, V. wodanis, and V. salmonicida, grow poorly at temperatures higher than 20°C. It is recommended to grow strains of these species at 15°C in Luria-Bertani broth (Difco) supplemented with 1 to 3% NaCl. Some Vibrio species, e.g., the V. halioticoli group and V. agarivorans, require addition of sodium alginate (0.5%) marine agar (343). Thiosulfate-citrate-bile salts agar (TCBS; Oxoid) is an ideal medium for the selective isolation and purification of vibrios. Strains which are able to use sucrose will form yellow colonies, while the others are green. So far this is the only proven selective medium for isolation of vibrios, although some isolates of Staphylococcus, Flavobacterium, Pseudoalteromonas, and Shewanella may present slight growth on it as well.

Most vibrios (except V. ezurae, V. gallicus, V. pectenicida, V. penaeicida, V. salmonicida, and V. tapetis) withstand the freeze-drying process very well. Coincidentally, these species are also difficult to grow on any culture media. Ampoules containing freeze-dried cultures prepared nearly 30 years ago have yielded viable and healthy colonies on tryptone soy agar. Normally, these ampoules are filled with 0.01 g of bacterial culture previously suspended in 0.5 ml of cryoprotectant mix (horse serum-D- glucose-nutrient broth-MilliQ water, 3:0.3:0.3:1). Alternatively, strains may be kept viable in Microbank vials, which contain 10% glycerol and porous beads, at –80°C for at least 5 years.

GENOTYPIC IDENTIFICATION

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An array of phenotypic and genomic techniques have become available for the identification of vibrios in the last three decades (97, 148, 296, 314, 315, 342, 402, 403). Ribotyping and PCR-based techniques, e.g., amplified fragment length polymorphism (AFLP), fluorescence in situ hybridization (FISH), amplified ribosomal DNA restriction analysis (ARDRA), random amplified polymorphic DNA (RAPD), repetitive extragenic palindromes (rep), and restriction fragment length polymorphism (RFLP), along with multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (MLST), have yielded the most valuable information about and new insights into the population structure of some species of the Vibrionaceae and have also provided a means of identifying these organisms. Below, we discuss some of the methods which have been most commonly used for the identification and typing of vibrios.

Jiang et al. (191, 192) discriminated V. cholerae serogroups O1 and O139 by using AFLP with the ApaI and TaqI restriction enzymes. They found that the genetic backgrounds of environmental and clinical V. cholerae strains are quite similar and concluded that pathogenic strains may in fact arise from nontoxigenic strains within the aquatic environment. Jiang et al. (191) demonstrated by AFLP analysis that the population structure of V. cholerae undergoes seasonal shifts. Certain clones are abundant in winter, and others are abundant in summer. More recently, Lan and Reeves (221) examined 45 V. cholerae isolates from the seventh pandemic and partitioned these isolates into 38 AFLP profiles. They concluded that AFLP is the best tool for discriminating clones from the seventh pandemic and suggested the design of PCR primers which target particular AFLP bands that could be used for epidemiological analysis through multiplex PCR or microarays analyses.

What do we know about the nonculturable vibrios? In 1982, Xu et al. showed that certain bacteria, e.g., V. cholerae, although metabolically active, were not able to grow on culture media. At that time, these authors already knew that environmental stresses (e.g., nutrient limitation or starvation and variations in pH, salinity, and temperature) could lead to such a state, for which they proposed the name "viable but nonculturable." Some researchers hypothesize that this is a "genetically programmed physiological response" to enable some bacteria to survive in the environment (258). Changes observed in VBNC bacteria include reduction of cell size, increase of cell wall thickness, decrease in the amount of RNA and DNA, and biofilm formation. Several Vibrio species, e.g., V. cholerae, V. shillonii, and V. vulnificus, can be VBNC and virulent. V. shillonii becomes VBNC when entering the cells of O. patagonica, but it remains metabolically active and multiples within its host (331, 333).

Several authors have recently shown that the most abundant prokaryotic groups, e.g., Archaea, cyanobacteria, the Cytophaga-Flavobacterium group, Roseobacter, SAR11, SAR86, SAR116, and SAR202 ( and/or ß proteobacteria), found in the marine environment are not readily culturable and that vibrios are rarely found in clone libraries from environmental samples (88, 131, 318). Although a high abundance of vibrios occurs in euthrophic coastal waters and in association with eukaryotes, these authors showed that vibrios represent only a minor fraction of the total bacterioplankton.

Heidelberg et al. (164), in a study of the bacterioplankton in the Chesapeake Bay, showed that proteobacteria compose up to 10% (3.1 x 10⁸ cells/liter) of the total bacteria, while Vibrio and Photobacterium compose up to 4% (2.1 x 10⁸ cells/liter) of the total bacteria. In the North Sea, vibrios accounted only for 10³ cells/ml (mainly particle associated) when genus-specific probes were used in FISH detection (103). The same authors found that by adding organic substrates (in micromolar concentrations) to the water, vibrios became dominant, reaching up to 65% (9.7 x 10⁵ cells/ml) of the total bacteria in a few hours (104). Vibrios not only could rapidly respond to nutrient-enrichment experiments but also maintained viability for up to 50 days under starved conditions. These authors concluded the high rRNA content of vibrios provide the potential for such rapid responses, which allow them to grow rapidly, outcompeting other members of the bacterial community. The increase in nutrient concentration in the water could lead to an increase in the size of the cells of vibrios, which in turn would escape predation by protozoans. Beardsley et al. (29) have indeed suggested that the low abundance of vibrios observed during certain periods and in some places may be the effect of massive selective grazing by heterothrophic nanoflagellates, which are abundantly found in aquatic environments.

The low fluorescence intensity of marine bacteria is one of the main drawbacks of FISH technology (103, 104). This is not really a problem for vibrios since these organisms have a high content of ribosomes. On the other hand, because several Vibrio species (e.g., V. harveyi, V. campbellii, V. rotiferianus, and other closely phylogenetic neighbours) have very similar 16S rRNA sequences, it may be difficult to perform reliable species identification.

Cho and Tiedje (68) successfully designed a microarray, containing up to 96 genomic fragments (about 1 kb long), for the identification of Pseudomonas species. The DNA chip designed showed good correlation with DNA-DNA homology measrurments. It was suggested that a chip containing 100,000 genomic fragments would allow the identification of most gram-negative bacteria (68).

rep-PCR was used to identify presumptive V. harveyi isolates responsible for luminous vibriosis in shrimp (139). These isolates had the major phenotypic features of the species V. harveyi (4, 5, 114, 170). They grew on TCBS agar, were motile, fermented glucose, were oxidase positive, and were sensitive to the vibriostatic agent 0/129 at 150 µg. Presumptive V. harveyi isolates were arginine dihydrolase negative and lysine and ornithine decarboxylase positive. Most isolates were luminescent and utilized D-gluconate, L-glutamate, D-glucuronate, heptanoate, D-galactose, and sucrose and grew at 40 °C, but they did not utilise L-histidine or L-arabinose. Most isolates (n = 31) clustered with the type strain of V. campbellii, LMG 11216^T. Because the isolates assigned to V. campbellii and to V. harveyi were very heterogeneous, DNA-DNA hybridizations were performed with representative strains to check the robustness of the clusters based on rep-PCR. The DNA- DNA hybridization experiments clearly showed that the presumptive V. harveyi isolates belong to the species V. campbellii, having at least 71% DNA similarity. In another study, rep-PCR was used to analyze the genomic diversity of vibrios isolated from the abalone gut (Haliotis spp.) (344). rep-PCR patterns using the primer GTG₅ showed that each abalone species has a particular population of vibrios which is related to V. halioticoli.

PHENOTYPIC IDENTIFICATION: THE PITFALLS OF CLASSICAL BIOCHEMICAL IDENTIFICATION AND DICHOTOMOUS KEYS

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Classical phenotypic identification techniques, including tests for arginine dihydrolase and lysine and ornithine decarboxylases, were among the most extensively used techniques to screen the diversity of Vibrio strains associated with marine animals and their habitat, and these tests have been proposed as reliable species identification schemes (4, 5, 12, 13, 39, 167, 222, 244, 297, 298). Variable results, e.g., for arginine dihydrolase of some species, have been reported, making their identification on this basis difficult (312). Biolog has been one of the most widely used phenotypic techniques for the identification of Vibrionaceae strains in the last decade (11, 13, 209, 266, 406). Bacterial strains are inoculated onto 95 different compounds which may serve as carbon sources. The identification of strains is based on the pattern of utilization of the 95 carbon sources. A very important diagnostic phenotypic feature for the identification of Vibrio species has always been the presence of flagella and thus motility (3). Nonmotile Vibrio species, e.g., the V. halioticoli group, have described (344, 346, 347), suggesting that the presence of flagella is not an essential diagnostic feature. Likewise, oxidase-negative V. metschnikovii and V. gazogenes strains have been documented, as have Vibrio strains that fail to grow on TCBS (4, 5). Colony variation is also a very common feature among Vibrio species, including the newly described species (12, 166, 425).

Fatty acids methyl ester (FAME) profiling was evaluated for the differentiation of Vibrionaceae species (36, 216, 299). FAME profiling is generally very useful as a chemotaxonomic marker, and apparently, differentiation at the genus level was possible. The similarity of FAME profiles among the different species examined was very striking, and the authors thus concluded that this technique could be used only as an additional phenotypic feature (36, 216). It became clear that the ample phenotypic variability within Vibrionaceae species pointed to the use of classification and identification scheme based on genomic data.

The phenotypic identification of genera and species of the Vibrionaceae is problematic. The main reason is the great variability of diagnostic phenotypic features, e.g., arginine dihydrolase and lysine and ornithine decarboxylases, susceptibility to the vibriostatic agent 0/139, flagellation, indole production, growth at different salinities and temperatures, and carbon utilization as revealed by Biolog (9, 13). Traditionally used as clear-cut tests for identification of species, the latter should thus be interpreted with greatest care. Dichotomous keys (see, e.g., references 4, 5,and 170) are misleading for the identification of Vibrionaceae isolates.

A comparison between a consensus molecular identification, including AFLP, DNA-DNA hybridization, and 16S rRNA sequences, on the one hand, and phenotypic identification (Biolog), on the other, shows that different Vibrio species appear within the same Biolog group. For instance, strains misidentified as V. harveyi by Biolog were later correctly identified as V. campbellii or classified as V. rotiferianus by AFLP and DNA-DNA hybridizations. Indeed, V. campbellii, V. harveyi, and V. rotiferianus have nearly indistinguishable phenotypes (136, 139). Strains misidentified by Biolog as V. campbellii turned out to be V. chagasii, while strains supposed to be V. splendidus were classified as V. kanaloaei. It was also remarked that many strains identified by AFLP as, e.g., V. cincinnatiensis, V. splendidus, and V. tubiashii, corresponded to multiple Biolog groups. Comparing AFLP and Biolog data, (i) a single genotype may correspond to a single phenotype (e.g., A8; V. brasiliensis), (ii) a single genotype may correspond to multiple phenotypes (e.g., A9; V. fortis), or (iii) multiple genotypes may be found in a single phenotype (e.g., A1, A2, A3, A4, and A5; V. coralliilyticus and V. neptunius) (388). It is nearly impossible to distinguish many of the currently known Vibrio species, e.g., V. splendidus-related species, solely on the basis of the phenotype (Table 2).

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The genera Vibrio and Photobacterium are among the oldest known bacterial genera (100, 201). The beginning of the taxonomy of vibrios can be traced back to the work of Pacini. Until the middle of the 1900s, the taxonomy of vibrios was dominated by morphological studies that tried to group strains on the basis of very few phenotypic features, e.g., flagellation, morphology, and curvature of the cells, and cultural aspects. These studies led to the description of many new Vibrio species. In the seventh edition of Bergey's Manual of Determinative Bacteriology (45), the genus Vibrio belonged to the family Spirillaceae and consisted of 34 species, which, with the exception of V. cholerae (listed as V. comma) and V. metschnikovii, were later reclassified into other genera, e.g., Campylobacter (C. fetus, C. jejuni, and C. sputorum), Comamonas (C. terrigena), or Pseudomonas (P. fluorescens) or no longer accepted as validly described species according to the Approved List of Bacterial Names (355). The genus Photobacterium, on the other hand, harbored one species, i.e., P. phosphoreum, and was allocated into the genus Bacterium of the family Bacteriaceae (45).

The heterogeneity within the genus Vibrio was highlighted by Davis and Park (90, 305). By examining morphological and biochemical features of most species of the genus Vibrio, they showed that it was quite artificial and concluded that at least three genera existed among the species examined. The foundation of modern Vibrio taxonomy was laid by a number of numerical (phenetic) and/or polyphasic taxonomic studies (17, 21-28, 74, 81, 125, 152, 229, 319, 393, 427, 438). Most of these studies clustered large collections of strains on the basis of their ability to utilize different (ca. 50 to 150) compounds as sources of carbon and/or energy, enzyme activity (e.g., gelatinase, chitinase, and DNase), salt tolerance, luminescence, growth at different temperatures, antibiograms, DNA base composition, morphological features, and other biochemical tests (e.g., oxidase, catalase, Voges-Proskauer, indole, nitrate reduction, arginine dihydrolase, and lysine and ornithine decarboxylases). The clusters defined by phenotypic features were further refined and validated by DNA-DNA hybridization experiments, and phenotypic clusters with about 80% similarity were found to correspond to DNA-DNA homology clusters with more than 80% similarity (24, 28). This suggests that for Vibrionaceae taxonomy, one should use 80% DNA-DNA similarity as the limit for species definition instead of the canonical 70% proposed by Wayne et al. (426).

In the eighth edition of Bergey's Manual of Determinative Bacteriology (50), the family Vibrionaceae, which was proposed by Véron, comprised Vibrio and Photobacterium along with Beneckea, Aeromonas, Plesiomonas, and Lucibacterium. The combination of Vibrio (V. anguillarum, V. cholerae, V. costicola, V. fischeri, and V. parahaemolyticus) and Photobacterium (P. mandapamensis [P. leiognathi] and P. phosphoreum) in a single family was an improvement in the taxonomy of these two related genera, which were thought for a long time to be only distantly related. Baumann et al. (22) proposed the genus Beneckea to encompass vibrios (i.e., B. campbellii, B. neptuna, B. nereida, and B. pelagia) isolated from the marine environment which required Na⁺ for growth. In subsequent studies, Baumann et al. (24-26) proposed that Beneckea species and Lucibacterium harveyi should be reallocated to the genus Vibrio, Aeromonas and Plesiomonas should be placed into other families, and V. costicola should be placed in another genus. These authors also suspected that the evolution of Vibrio and Photobacterium species was driven mainly by vertical processes (mutations) rather than horizontal gene transfer. The DNA-DNA relatedness studies among Vibrio and Photobacterium species underpinned the taxonomy of these groups (26, 27, 319). These studies disclosed a core group of related vibrios, i.e., the V. harveyi group, consisting of V. harveyi, V. campbellii, V. natriegens, V. alginolyticus, and V. parahaemolyticus. V. harveyi and V. campbellii were found to have 61 to 74% DNA-DNA similarity, while V. parahaemolyticus and V. alginolyticus had 61 to 67% similarity. Reichelt et al. (319) also proposed biotypes I and II for V. splendidus and V. pelagius, but they suspected that these biotypes could be different species. Biotypes I and II of V. splendidus and V. pelagius showed a maximum of 61 and 58% DNA-DNA similarity, respectively. Additionally, the biotypes of both species were clearly distinguishable by phenotypic features. Nevertheless, researchers are still using the biotype designation today (368). Arias et al. (7, 8, 111) have suggested that the two biotypes of V. vulnificus should be abolished. These biotypes should be considered as different species according to the current species definition (361).

Baumann et al. (24-26) compared the amino acid sequence differences of glutamine synthetase, superoxide dismutase, and alkaline phosphatase to distinguish Vibrionaceae species. Because the determination of amino acid sequences was very time- consuming and cumbersome at that time, Baumann and colleagues applied a technique called microcomplement fixation, which is based on the immunological reaction of antigens and antisera of the target proteins. On the basis of this analysis, they concluded that Beneckea species, P. fischeri, and P. logei should be transferred to the genus Vibrio (25) (see also the first edition of Bergey's Manual of Systematic Bacteriology [210]). They also mentioned that they applied a certain "subjective judgement" about the limits of the genus Vibrio because they found that this genus was highly diverse. Several species, e.g., V. cholerae, V. fischeri, V. logei, and V. costicola (now Salinivibrio costicola), were distantly related to each other and to the Beneckea species.

PHYLOGENY OF THE VIBRIOS

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In the last two decades, bacterial phylogeny has been enriched with chronometers, e.g., rRNAs (5S, 16S, and 23S), to reconstruct bacterial phylogenies but also to be used as taxonomic markers for identification. In many cases, the phylogenies obtained by 16S rRNA sequencing pointed out the inadequacy of grouping bacteria by the classical criteria, e.g., morphology and biochemical features. The close rel