PATHOGENS: TYPE OF ILLNESS AND CHARACTERISTICS OF THE ORGANISMS

V. cholerae

This species was fi rst described as the causative agent of epidemic cholera disease by Pacini in 1854 and later by Koch in 1883, and it remains one of the deadliest bacterial disease agents worldwide (Lipp et al., 2002). Although cholera epidemics have been essentially eliminated in the United States, the disease is still endemic in parts of Asia and Africa and more recently South and Central America. The global inci-dence of this disease remains devastatingly high:

approximately 100,000 to 300,000 cases are reported annually worldwide to the WHO, which estimates that these fi gures probably refl ect only 1% of the actual disease incidence (Zuckerman et al., 2007).

Nonepidemic V. cholerae is also responsible for occa-sional seafood-borne disease in the United States and can be distinguished from epidemic disease by the severity of symptoms, the serogroups of associated strains, and the capacity for global spread.

Anita C. Wright and Keith R. Schneider • University of Florida, Food Science and Human Nutrition Department, 359 FSHN Bldg., Newell Drive, Gainesville, FL 32611.

enters the cell and transverses across the cytoplasm, releasing the A subunit into the cytoplasm. Activity of the A subunit blocks the host machinery for recycling the intracellular signaling molecule, cyclic AMP. Spe-cifi cally, the toxin transfers ADP ribose to G proteins that are involved in this process. The net result is the permanent activation of host adenylate cyclase, which causes signifi cant increases in cyclic AMP levels, lead-ing to secretion of massive amounts of chloride ions into the lumen of the intestine. Subsequent osmotic imbalance induces dramatic water loss from the cells and tissues, resulting in characteristic voluminous diar-rhea. This rapid loss of large fl uid volumes accounts for the severity and high mortality associated with cholera. V. cholerae bacteria lacking CT are not able to cause epidemic disease (Morris, 2003).

Other virulence factors include the toxin coregu-lated pilus (TCP) that is expressed during coloniza-tion of the small intestine (Taylor et al., 1987a).

Interestingly, this pilus also serves as the receptor for a lysogenic fi lamentous bacteriophage (CTX) that carries the genetic material for the production of CT (Waldor and Mekalanos, 1996). Nontoxigenic V. cholerae strains are converted to toxigenic, poten-tially epidemic, strains following infection by this phage. Genes for TCP and CTX are both regulated by the same transcriptional activator, ToxT (DiRita et al., 1991). Motility is also required for virulence (Richardson, 1991), and chemotaxis is important for colonization and induction of CT expression in vivo (Lee et al., 2001). Other possible virulence factors include outer membrane proteins or porins (Proven-zano and Klose, 2000), and an inner membrane pro-tein ToxR that is produced by all Vibrio species and coordinately regulates the other virulence factors (Miller and Mekalanos, 1984).

Treatment of cholera includes oral or intravenous rehydration and antibiotic therapy. V. cholerae is sen-sitive to a variety of antibiotics, especially tetracyclines, but multiply drug-resistant strains have been reported (Morris, 2003). Vaccines offer protection against dis-ease but are expensive to produce and deliver, and the immunity provided may be limited (Taylor et al., 1988;

Zuckerman et al., 2007; Ford et al., 2007). Genetically Epidemic cholera is characterized by massive

watery diarrhea, which produces rapid fl uid loss that, if untreated, leads to frequently fatal dehydration.

The disease requires a relatively large infectious dose, estimated to be about 1 million bacteria, and suscep-tibility to the disease varies widely among healthy adults (Sack et al., 1972). Following ingestion, the bacterium multiplies in the lumen of the small intes-tine and uses pili to anchor to enterocytes in the gut (Taylor et al., 1987b). The primary virulence factor is the cholera toxin (CT) that is secreted from the bac-terium during infection and is responsible for the characteristic diarrhea (Finkelstein and LoSpalluto, 1969). The activity of CT has been studied exten-sively, and it serves as a model for the study of other bacterial toxins with similar structures (Sandvig and van Deurs, 2002; Lyerly et al., 1982).

As shown in Figure 2, CT is comprised of two protein subunits designated A and B, and these pro-teins combine to make the active holotoxin (Kaper et al., 1995). The single A subunit has the enzymatic activity responsible for symptoms and is surrounded by fi ve B subunits that provide the binding capacity of the toxin. After docking to the intestinal cells, the toxin

Figure 1. V. vulnifi cus bacteria that are attached to diatom are in-dicated by arrow. (Figure provided by Maria Chatzidaki-Livanis.)

Table 1. Typical symptoms associated with different Vibrio species

Vibrio species (type) Symptoms

Gastroenteritis Severe diarrhea Septicemia Wounds

V. cholerae (epidemic) Less common Common No Rare

V. cholerae (nonepidemic) Common No Rare Rare

V. parahaemolyticus Common No Rare Less common

V. vulnifi cus Less common No Common Common

deletion of the gene (tdh) encoding TDH corresponds to loss of symptoms in animal models (Nishibuchi et al., 1992). Diarrhea may result from toxin-mediated induction of Ca^2-activated chloride channels, lead-ing to fl uid accumulation (Baffone et al., 2005). Toxic molecules related to type three secretion systems have also been implicated in disease and are under investi-gation (Liverman et al., 2007).

Treatment for V. parahaemolyticus involves rehydration, especially for persistent diarrhea, and infected persons may also benefi t from antibiotic therapy with tetracyclines or quinolones (Morris, 2003). A vaccine is not available for V. parahaemo-lyticus, and vaccine development seems unlikely due to the typically mild nature of symptoms. However, recent increases in the global spread of epidemic V. parahaemolyticus disease and better understand-ing of virulence mechanisms may lead to improved prevention strategies.

V. vulnifi cus

V. vulnifi cus was fi rst described by Hollis et al.

(1976), and typical symptoms are dramatically differ-ent from those of other pathogenic vibrios. Disease can result either as a consequence of seafood con-sumption or from exposure of wounds to seawater or through the handling of seafood (Blake et al., 1979).

Disease incidence is relatively rare compared to the engineered, live attenuated oral vaccines hold great

promise and generally involve deletion of the gene (ctxA) encoding the enzymatic A subunit of CT (Kaper et al., 1995). Unfortunately, these vaccine strains still retain some reactogenicity, and emerging strains can be antigenically different from vaccine strains, neces-sitating the construction of new vaccines.

V. parahaemolyticus

V. parahaemolyticus was fi rst described as the causative agent of outbreaks of gastrointestinal ill-ness in Japan (Fujino, 1953). Symptoms associated with these infections contrast greatly with epidemic cholera and generally present as mild gastroenteritis with watery diarrhea that resolves without treat-ment. Occasional dysentery, wound infections, and septicemia are also caused by V. parahaemolyticus, but fatalities are rare and generally follow septicemia (Su and Liu, 2007). V. parahaemolyticus produces several hemolytic toxins. The fi rst described toxin was the thermal stable direct hemolysin or TDH, whose activity is characterized by the Kanagawa phenomenon that is based on lysis of human red blood cells on Wagatsuma agar (Miyamoto et al., 1969). Some clinical isolates produce a genetically similar toxin called the TDH-related hemolysin (TRH), and other strains produce both TDH and TRH (Honda et al., 1988). Experimental genetic

Figure 2. Activity of CT. CT B subunits bind host intestinal cells, followed by endocytosis and release of the A subunit (black circle), which ribosylates G proteins to activate adenylate cyclase. The subsequent increase in cyclic AMP (cAMP) results in protein phosphorylation, extrusion of chloride ions, and massive diarrhea. (Adapted from Kaper et al., 1995.)

V. vulnifi cus disease. For example, excess physiologi-cal iron as a consequence of hereditary hemochroma-tosis increases susceptibility to disease (Blake et al., 1979). Wright et al. (1981) found that exogenously administered iron in mice dramatically reduced the 50% lethal dose from about 10,000 to 10 bacteria.

Production of bacterial iron-sequestering siderophores also contributes signifi cantly to virulence (Litwin et al., 1996). Other reported virulence factors include fl a-gella (Lee et al., 2006; Kim and Rhee, 2003), pili (Paranjpye and Strom, 2005; Paranjpye et al., 2007), and quorum sensing (Kim et al., 2003), as well as numerous secreted molecules (proteases, phospholi-pases, DNases, etc.) whose relevance to virulence is unclear, as reviewed by Gulig et al. (2005). V. vulnifi -cus hemolysin is unrelated to the hemolysins of V. para-haemolyticus but shows genetic similarity to those found in V. cholerae and other Vibrio species (Yama-moto et al., 1990). Furthermore, the V. vulnifi cus hemolysin is unlikely to be a virulence factor because inactivation of the vvhA gene encoding the hemolysin does not reduce virulence in mice (Wright and Morris, 1991; Fan et al., 2001). Another hemolysin, referred to as repeats in toxin or RTX, has been implicated in disease in animal models (Lee et al., 2007).

Encapsulation by polysaccharide is a primary virulence factor and contributes to the ability of V. vulnifi cus to cause systemic disease by providing protection from the lytic activity of serum and from phagocytosis during systemic disease (Tamplin et al., 1985; Yoshida et al., 1985; Simpson et al., 1987).

Encapsulation is marked by formation of opaque colonies; however, this phenotype may undergo spon-taneous phase variation to an alternate colony type, which appears more translucent and has reduced capsular polysaccharide (CPS) expression and thus decreased virulence in animal models (Yoshida et al., 1985; Amako et al., 1984). The different colony types are shown in Figure 4 and have been observed with other Vibrio species. A CPS operon was recently described for V. vulnifi cus and showed genetic similarity to the Esherichia coli group 1 capsule (Chatzidaki-Livanis et al., 2006b; Chatzidaki-Livanis et al., 2006a; Wright et al., 2001). Multiple types of translucent-phase variants have been identifi ed with differing degrees of encapsulation and variation in the genetic organization of the CPS operon. Acapsu-lar variants show deletion mutations in this operon and are locked in the translucent phase, while strains with partial CPS expression retain an intact operon and reversible phase variation.

V. vulnifi cus septicemia is rapidly fatal, and the early administration of antibiotics is critical to the outcome of disease (Klontz et al., 1988). Combina-tions of antimicrobials may be more therapeutic and other vibrio pathogens, and only 30 to 60 cases are

reported annually in the United States (Feldhusen, 2000). However, this single species is responsible for nearly all of the deaths related to bacterial contami-nation of seafood in the United States. The mortality rate is exceptionally high ( 50%), and risk of disease may be greater in countries outside the United States, where increased exposure of consumers to raw sea-food and lower immune status is more common (Park et al., 1991). A recent survey in Japan detected six cases of V. vulnifi cus septicemia during a 20-day period (Ono, 2001).

Unlike the other vibrio pathogens, illnesses caused by V. vulnifi cus are rarely seen in healthy adults or children. This species is very much an opportunistic pathogen, and persons who are at risk for this disease generally exhibit some type of under-lying condition that includes alcoholic cirrhosis, hepatitis C, diabetes, hemochromatosis (iron over-load), and immune system dysfunction (Hlady and Klontz, 1996; CDC, 2005). In these persons, a ful-minating, primary septicemia can occur within hours of exposure and produce symptoms that are similar to other types of gram-negative toxic shock, includ-ing rapid drop in blood pressure, intravascular coag-ulation, and multiple organ failure. A distinguishing characteristic of V. vulnifi cus sepsis is the appearance of bollous lesions on the extremities, as shown in Figure 3. V. vulnifi cus also causes gastrointestinal ill-ness with mild diarrhea and vomiting. This organism is the most common cause of serious wound tions associated with Vibrio species, and these infec-tions may result from exposure of breached skin surface to seawater or contaminated seafood han-dling (Shapiro et al., 1998; Howard and Lieb, 1988) (reviewed by Oliver [2005]). In compromised hosts, wound infections may also lead to septic shock and death.

Experimental evidence supports epidemiological data indicating that host iron status contributes to

Figure 3. Bollous lesions associated with V. vulnifi cus septicemia.

Specifi c physical parameters greatly infl uence the environmental distribution of the different pathogenic Vibrio species; however, responses to these parameters vary somewhat among species.

These bacteria grow optimally at temperatures between 22 and 42C within a pH range of 5 to 11 for salinities ranging from 1 to 8% (Kaspar and Tamplin, 1993; Motes et al., 1998; Kelly and Stroh, 1988; Kelly, 1982). In general, the numbers of all Vibrio species peak in warmer months and decline after extended exposure to cold temperatures, but tolerances to extremes in temperature and salinity vary with species. V. parahaemolyticus can be recov-ered from colder, more saline environments that generally do not support survival of V. vulnifi cus or V. cholerae (Kaneko and Colwell, 1973; Kelly and Stroh, 1988; Kelly, 1982). Thus, extremes in tem-perature and salinity may limit distribution of a par-ticular Vibrio species in shellfi sh. It should also be noted that combined parameters of temperature, pH, and salinity have interactive effects on growth and survival and vary with depth. Additional bio-logical factors that are likely to infl uence the preva-lence of these species in the environment include fl uctuations in nutrient availability, algal blooms, competition with other bacterial fl ora, ultraviolet irradiation, and availability of invertebrate or verte-brate hosts.

V. cholerae

Since 1817 there have been at least seven pan-demics of V. cholerae that have spread throughout the world (Lipp et al., 2002). In 1849 John Snow famously traced the source of a cholera outbreak in London to a tainted well, laying the foundations for the fi eld of epidemiology and establishing principles of modern public health. In cholera-endemic regions of the world, V. cholerae is transmitted primarily through contaminated drinking water. Both natural and human-made disasters exacerbate outbreaks of cholera due to a breakdown of infrastructure for sew-age disposal and water treatment and to delay of medical treatment. Although cultural taboos often prohibit the consumption of shellfi sh in regions where cholera has been endemic historically, more recent epidemics in South America clearly implicate raw or undercooked seafood and shellfi sh. Sporadic cases reported in New Jersey and Florida were attributed to imported blue crab (CDC, 1991; Tauxe et al., 1995). Improved understanding of the biology of Vibrio species supports the essential link of this bac-terium to the aquatic environment as the primary res-ervoir of disease (Colwell et al., 1977). V. cholerae has a relatively low salinity requirement compared to increase survival, and minocycline combined with

cefotzxime or fl uoroquinolone is recommended (Chuang et al., 1998). A vaccine against V. vulnifi cus is not available.

ENVIRONMENTAL SOURCES: INCIDENCE