Vibrio vulnificus

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A Microbial Biorealm page on the genus Vibrio vulnificus

Classification

Scanning electron micrograph (SEM) depicts a grouping of Vibrio vulnificus bacteria; Mag. 13184x. Photograph courtesy of CDC by Janice Carr and colorized by James Gathany.

Higher order taxa

Bacteria; Proteobacteria; Gammaproteobacteria; Vibrionales; Vibrionaceae; Vibrio

Genus and Species

Vibrio vulnificus

Nomenclature

In 1976, John L. Reichelt designated V. vulnificus as a new pathogenetic species under the genus Beneckea.(15) Three years later, J.J. Farmer proposed the pathogen be moved to the genus Vibrio, basing his argument on its genetic relatedness and phenotypic similarities to other species within Vibrio. Approved, the microbe received the scientific name Vibrio (“Beneckea”) vulnificus.(14)

Description and significance

V. vulnificus, a Gram-negative, rod shaped, halophile, is a virulent bacterium associated with about 95 percent of all seafood related deaths.(7) It is a human pathogen known to cause gastroenteritis, which is an infection of the stomach and intestines, wound infections and primary septicemia, which occurs when the bacterium enters the host's blood and causes infection throughout the body.

This organism can typically be found in salty, coastal waters, thriving especially in molluscan shellfish including oysters and clams, thereby causing health risks to those who ingest raw or undercooked seafood.

V. vulnificus has three biotypes, with biotype one, identified as a new species in 1976, causing the greatest public health concern due to its involvement with human disease.(7) Furthermore, biotype two is associated with infected eels, and biotype three is associated with wound infections of humans.(5)

Genome structure

The genomes of two strains of V. vulnificus, YJ016 and CMCP6, have been fully sequenced, each with two, circular chromosomes.(6)

Chromosome I of strain YJ016 has a length of 3,354,505 nucleotides, while chromosome II contains 1,857,073 nucleotides, with a total size of 5,260,086 base pairs.(4)

Chromosome I of strain CMCP6 has 3,281,945 nucleotides in length, while chromosome II has 1,844,853 nucleotides, for a total size of 5,126,798 base pairs.(4)

It is believed that Vibrios have two chromosomes as an evolutionary advantage. The larger chromosome plays a role in growth, while the smaller one is involved in adaptation and environmental change.(7) Phenotypic variation is also present within V. vulnificus. It is observed that no two strains are genetically identical.

Cell structure and metabolism

Vibrio vulnificus pili is used for many reasons such as motility, adherence and secretion. Courtesy of Rohinee Paranjpye and Mark Strom, Northwest Fisheries Science Center, NOAA Fisheries Service, Seattle, WA

V. vulnificus is a Gram-negative bacterium, which contains an outer membrane which surrounds a thin layer of petidoglycan, or cell wall, and an inner membrane. It contains pili- hairlike appendages found on the surface of the bacteria- for motility, adherence to epithelial cells and involvement in biofilm formation. Some strains contain capsules which determine virulence and are also associated with biofilm formation.(3) In addition, iron is an important factor for growth and siderophores are produced, which are receptors that bind and deliver the iron to the bacteria.(1)

This organism is also known to be “lactose-positive” with its fermentation of lactose, salicin and cellobiose. But since phenotypic variation is observed, about 15% of strains were found to be lactose negative and another 15% were found to be sucrose positive.(7)

In addition, this bacterium is a known halophile, and usually grows in areas with a minimum of 5% salinity.(7)

Lipopolysaccharides can be found on the outer layer of the membrane, and contain endotoxin.(5) This trait is the major cause for the organism’s virulence and is the leading factor which causes shock or death in an infected individual.

Ecology

V. vulnificus resides in marine environments where temperatures are warmer: usually in tropical, subtropical, and temperate regions. This includes both the Atlantic and Pacific oceans, and coastal, estuarine, and marine ecosystems.(11) V. vulnificus’s range of temperature for growth begins at 13°C, with the optimal growth temperature between 20°C and 30°C.(16)(17) V. vulnificus survives in low to moderate salinities, with an optimal range from 15 to 25 ppt.(24) However, it can also be tolerant of higher salinities (30, 35, and 38 ppt) when temperature is at an optimal level, such as 21°C.(16)

When water temperatures drop, specifically at or below 13°C, V. vulnificus is able to go into a dormant state, titled a “viable but nonculturable state” (VBNC). In this state, V. vulnificus is able to survive otherwise detrimental environment stresses through turning off specific genes. While in this form the microbe is unable to be cultured with conventional media. When water temperatures rise again, such as in spring, V. vulnificus is becomes metabolically active and culturable again. (12)

Another survivability factor V. vulnificus uses is a generalized stress response.(5) Depending on its environment, changes induce production of heat shock proteins in response to different temperatures, changes in osmolarity, the presence of pollutants, or its interactions with other organisms. With the production of these proteins, the bacteria may adapt comfortably in its environment.

V. vulnificus can be found both suspended in the water column, within sediment, or within ocean fauna, such as shrimp, oysters, and clams. V. vulnificus are able to attach to particulates or organisms in the water column, making them more susceptible to accumulation within filter-feeders. In oysters alone, concentrations of V. vulnificus are 100 times greater than that of the surrounding waters. As oysters are usually consumed raw on the half shell, there is a high infection risk. (12)(13) Interestingly, with such a high concentration, the actual occurrence of infection is much lower than expected. This could be accounted for virulence differences in multiple biotypes living in oysters, or unreported infection that only results in gastroenteritis and not hospitalization.

A potential concern is whether rising global ocean temperatures will increase the rate of V. vulnificus infection. Research has found a recent growth in V. vulnificus’s geographic range and an increase in infection occurrence. However, the degree of this rise is still undergoing research, with hopes for a better understanding of how climate change will impact the pathogen and by extension, humans.(22)

Pathology

Multiple factors allow V. vulnificus to be successful as a pathogen.(23) There are three main biotypes distinguished by phenotypic and host differences. Biotype one is primarily a human pathogen.(20) It is the leading cause for the majority of all reported seafood-related deaths (95% in the US alone). Death is caused by infection of the bloodstream leading to primary septicemia.(11) Biotype 2 is associated exclusively with eels, causing eel vibriosis or death. This type differs from biotype one in multiple ways including the production of exoproteins and the O-antigenic side chain on the lipopolysaccharide molecule.(7) Biotype 3 is genetically a hybrid of 1 and 2. It is of increasing concern, having correlations between infected tilapia and wound infection in Israel.

There has been little success in finding an avirulent strain with its own distinguishing factor. However, differences in degrees of virulence have been found in two major genotypes of V. vulnificus. Differences in virulence-correlated gene (vcg) locus coincide with the origin of V. vulnificus isolates. Strains taken from clinical origin (vcgC) are associated with a significantly higher virulence then those of environmental origin (vcgE).(23) This variance may account for the significant discrepancy between the high level of infection risk and the low level of actual infection within humans.(13)

This bacterium may infect an individual via entry through ingestion or through a wound infection. Some symptoms of infection depend on which way V. vulnificus enters the bloodstream, but primary septicemia can occur via either infection. (13)

Ingestion

V. vulnificus (Biotype 1) can enter a host’s body via ingestion, the most common method being through consumption of raw oysters. A healthy person will experience mild to moderate gastroenteritis. However, those with an underlying immune disorder are at greater risk. One common example of such is chronic liver disease. One survey, dating between 1988 and 1996 and located around the US Gulf Coast, found that 80% of those with primary septicemia from V. vulnificus infection also had a liver disease.(24)For ingestion-specific infection, symptoms can include diarrhea, abdominal cramps, nausea, vomiting, head-aches, chills, and low-grade fever. Infection can also result in primary septicemia with symptoms including severe hypotension and secondary lesions on extremities. (12) Incubation rates are quick with symptoms occurring after 24 hours. In this time, V. vulnificus enters the bloodstream from the gastrointestinal system, infecting the host. Antibiotic treatment is critical in this small window of time, as mortality from infection can occur in a 24 to 72 hour period.(13)(12)

V. vulnificus has a significant number of potential determinants of virulence. Via the ingestion route, V. vulnificus can pass through the low pH of the stomach an acid neutralizing agent. After passing through the upper gastrointestinal system, V. vulnificus infects the host’s bloodstream by penetrating the intestinal wall. It then works against the innate immune system and complement activity. How V. vulnificus is successful in its attack is debated, but one popular hypothesis is that the microbe is able to induce macrophage apoptosis of lymphocytes in the blood.(13) Besides this, other attacking strategies include the presence of a capsule to avoid host phagocytosis, biofilm formation, and pili for attaching to host cells and iron transfer.(3) The bacterium may cause cell death through the use of lipopolysaccharides (LSP) in its cell wall. LSP is a common endotoxin found in numerous bacterial pathogens, including V. vulnificus.(11) LSP in the cell wall may contribute to the induction of inflammation, tissue damage, and shock during infection via V. vulnificus.

In addition to these defenses by V. vulnificus, it also produces an exotoxin, hemolysin (VvhA) that facilitates iron release from hemoglobin within the bloodstream. Tissues may be affected via increased vascular permeability, apoptosis of endothelial cells, and increased nitric oxide production. The exotoxin mostly causes cell death by pore formation in the cell membrane. Its level of importance in the infection process is still in discussion.(20)

Wound Infection

Wound infection, similar to entry via ingestion, can lead to primary septicemia. However, entry of V.vulnificus to the bloodstream is through an open wound coming in contact with contaminated water or a contaminated host. Hosts include organisms from marine or brackish environments, including oysters. (19) For example, shucking oysters can result in ungloved hands being cut open, with wound entry from the contaminated oyster. However, recent research has found that V. vulnificus entry was carried over from tilapia, a mainly freshwater fish, within Israel (20). There is necrosis and large bullous skin lesions around the infected wound.(19) While visually more displeasing, wound infection is usually less severe and the survival rate is greater. The fatality rate from wound infection usually is 20 – 30 % lower than that of ingestion infection

Treatment

Antibiotics commonly used against V. vulnificus include a wide variety of different antibiotics. The most effective is tetracycline in a combination with gentamicin or chloraphenicol. This is relative to both ingestion and exposure. For serious soft-tissue wound infections, antibiotic therapy rarely is sufficient on its own and surgical debridement is essential.(20)(19)

Prevention

V. vulnificus can be killed by freezing or boiling. It is always best to consume fully cooked seafood. Those who are at greater risk of mortality from infection (such as those with chronic liver disease) should avoid consumption of raw or undercooked shellfish. Precautions have been made throughout the world to educate the public of the risks. For example, in the U.S., warning labels on seafood products were required that indicated the risk of infection. A ban of oyster imports from the Gulf of Mexico is also in place between the 1st of April and the 1st of October, where ocean temperatures have risen to levels of greater risk.(21)

Application to Biotechnology

This microbe does not produce any antibiotics, but does produce many enzymes and cytoxins. Some of these enzymes and cytotoxins include proteases, endotoxin, lipopolysaccharides, hemolysin, lipases, DNAases and cytolysins.(1) All of these are known virulence factors, contributing to gastroenteritis, septicemia and infection of wounds.

Furthermore, this microbe produces additional sigma factors, or initiation factors for prokaryotic transcription. One in particular, called RpoS, demonstrates a role in regulation to environmental stresses.(7) Its mutant shows a decrease in motility and a decrease in production of a couple of virulence factors. The production of cyclic AMP and a mutant in the cya gene shows similar results in inhibition and decrease production or activity.(7)

Current Research

Since it is known that V. vulnificus thrives in saline conditions, some researchers have conducted experiments to observe the result of exposing the V. vulnificus YJ03 strain in low-salinity environments. Results show that a strain adapted to acid, bile and heat stresses, did not survive the exposure to low salinity. On the other hand, strains of V. vulnificus that were adapted to bile stresses and were in the state of exponential and stationary growth, where cross protected from low-salinity.(8)

When it comes to food contamination, research has been done to find a way to detect different kinds of pathogens, even if they are present in very low concentrations. An assay found to detect these pathogens, including V. vulnificus, is called buoyant density gradient centrifugation, quickly separating the pathogen from food matrices. After the centrifugation, the next procedure is PCR (polymerase chain reaction) to detect bacterial DNA that could be present even in minute concentrations.(10)

Other researches have investigated ways to inhibit or prevent V. vulnificus growth in oysters. They observed the effects of using electrolyzed oxidizing (EO) water, acting as an antibacterial against V. vulnificus. Within seconds of exposing the oysters to the EO water, a decrease in the bacterium population occurred. Further experimentation showed that exposure to EO water, containing high amounts of chlorine, for more than 12 hours, were harmful to the oyster. They observed that the optimal exposure time ranged between 4 to 6 hours, without being hazardous to the oyster. In light of this discovery, they suggested that this treatment be used after harvest to reduce contamination of the seafood.(9)