Vibrio vulnificus

1. Vibriovulnificus Disease and pathogenesis

2. V. vulnificusis a halophilic marine bacterium. As othermembersof the genusVibrio, family Vibrionaceae, itis a Gram-negative rod, aerobic and facultativelyanaerobic, motilebymeansof a polarsheathedflagellum, and isoxidase and catalasepositive.

3. V. vulnificuspossesses a wide array of virulence factors, including acid neutralization, capsular polysaccharide expression, iron acquisition, cytotoxicity, motility, and expression of proteins involved in attachment and adhesion.

4. Biotypes Strains of V. vulnificusare classified into biotypes based on their biochemical characteristics. Strains belonging to biotype 1 are responsible for the majority of human infections, while biotype 2 strains are primarily eel pathogens. A recently identified third biotype was shown to possess biochemical properties of both biotypes 1 and 2 and to cause human wound infection.

5. Genotypes The purpose of genotyping analysis is to identify genetic indicators of virulence. One such method employed random amplified polymorphic DNAPCR to identify an amplicon present primarily in clinical isolates. Subsequent analysis of this virulence-correlated gene (vcg) revealed consistent sequence variations that correlate highly with the isolation source. Strains possessing the sequence common among clinical isolates were designated C type, while strains possessing the environmentally correlated sequence were designated E type.

6. Clinical and environmental strains can also be differentiated by comparing 16S rRNA sequences. Typing methods have been employed to examine the environmental distribution of potentially virulens strains of V. vulnificus. Examining the presence of the C and E genotypes in oysters and surrounding seawater leads to the result that while these genotypes are present in approximately equal proportions in water, there is a large preponderance of the potentially less virulent E types in oysters.

7. Host Resistance Acid resistance Since disease typically results from raw shellfish consumption, the highly acidic gastric environment is one of the first host defenses encountered by this bacterium. A common method employed by gram-negative bacteria to neutralize low pH environments is through the breakdown of amino acids to yield amines and CO2 and V. vulnificus appears to use similar system when it encounters acidic environments in vitro. Values of acid tolerance at pH 3.5 (HCl) for three strains of V. vulnificuspreviously acid adapted for different times in HCl at pH 5. Bars represent the standard error from three replications.

8. Host immune response Analysis of cytokine activation during V. vulnificusdisease has shown that several proinflammatory cytokines, are specifically induced and predominantly expressed during infection. This kind of reaction will lead to the septic shock, which is a hallmark of V. vulnificus disease. Moreover, during infection this bacterium reduces the number of lymphocytes through apoptotic activity, but neutrophil numbers do not decrease, indicating that other leukocytes are being targeted. Specific examination of macrophages revealed that clinical strains of V. vulnificuswere able to induce macrophage apoptosis. CPS V. Vulnificusprimarily achieves the evasion ,from host immune defense, through surface expression of CPS. The presence of capsule provides resistance to opsonization by complement and thus avoidance of phagocytosis by macrophages.

9. Iron acquisition Another feature associated with V. vulnificusdisease in elevated serum iron levels in infected individuals. It’s possible to see the importance of iron acquisition related to pathogen’s virulence in the differential survival of the C and E genotypes in human serum. Precisely how excess serum iron confers an advantage to V. vulnificusis still unclear. However, two theories have been put forth:

10. Cellular damage and cytotoxicity • In V. vulnificus, an extracellular hemolysin encoded by vvhA contributes to iron release through its hemolytic activity and is also responsible for the bacterium’s cytotoxic activity. • Pathological effects are: • Tissue necrosis; • Fluid accumulation; • Intestinal irregularities; • Partial paralysis; • Lethality… • Another enzyme suggested to be involved in V. vulnificusvirulence is an extracellular protease, designated VvpE, which is nonspecific and has broad substrate specificity. This enzyme is able to cause: • Tissue necrosis; • Edema; • Vascular permeability; • Degradation of type IV collagen; • Activation of procaspase-3. • Endotoxic shock is also characteristic of V. vulnificusdisease, and this symptom is thought to be due to the presence of lipopolysaccharide. VvhA • VvpE LPS

11. Attachment and motility For V. vulnificusthe cell-cell contact is required for citotoxicity, in fact pili are used by many gram-negative bacteria for adherence to host cells. In addition to adhesion, flagellum-based motility is important for a variety of bacterial processes including biofilm formation and pathogenesis. In V. vulnificus case, the flagellum is necessary to virulence and cytotoxin delivery. Pili Flagella

12. Virulence regulation Quorum sensing V. Vulnificusrequires coordinated expression of a variety of virulence factors in order for tissue destruction and lethality to occur. Therefore, the bacterium likely employs global regulators to control its pathogenesis. QS through the LuxS/LuxR-type system is characteristic of this pathogen. LuxS is responsible for synthesis of autoinducer 2 and LuxR is the associated transcriptional regulator. Both of those gene influence the bacterial cytotoxicity; in particular luxS influences also the transcription of vvhA and vvpE, leading to increasing and decreasing of these toxins, respectively. V. VulnificusQS system also influences gene expression of macrophages and potentially contribute to survival of the bacterium during the infection. The presence of cyclic-di-GMP, which is a small molecule involved in signalling pathways, allows bacteria to sense and adapt to their environmental surroundings and to regulate their virulence.

13. So… Which are the consequences of Vibriovulnificus infection?

14. Vibriovulnificusis an opportunistic and highly lethal human pathogen. This bacterium occours naturally in marine and estuarine waters throughout the world. It thrives in warm waters and it is therefore common in tropical and subtropical estuarine and sea waters.

15. V. Vulnificusis a part of the natural flora of coastal marine environments worldwide and has been isolated from water, sediments, and a variety of seafood, including shrimp, fish, oysters, and clams.

16. Infections generally occurs from March to December with peaks from May to October depending on water temperature. SeasonalityofVibriovulnificus

17. Consumption of seafood containing V. vulnificuscan result in a severe , fulminant systemic infection. Characteristics of this disease include fever, chills, nausea, hypotensive septic shock, and the formation of secondary lesions on the extremities of patients. In addition to septicemia, V. vulnificus can produce serious wound infections that typically result from exposure of open wounds to water harboring the bacterium. Like systemic disease, wound infections progress rapidly to cellulitis, ecchymoses, and bullae, which can progress to necrotizing fascitiis at the site of infection. Characteristic skin lesions associated with Vibriovulnificus infection on the leg in a 75-year-old patient with liver cirrhosis in whom septic shock and bacteremia developed. B V. vulnificusbacteremia developed one day after a fish bone injury on the fourth finger of the left hand (arrow) in a 45-year-old patient with uremia.  C. Gram-negative curved bacilli isolated from a blood sample of the 45-year-old patient with uremia.

18. For the majority of the people, the bacterium is harmless however, people with chronic liver diseases including: • hepatitis, • cirrhosis, • hemochromatosis (iron storage disease), • liver cancer; • and people with chronic diseases such as: • Diabetes, • Chronic kidney disease, • Conditions impairing immune system; • are at higher risk of serious infection. • But…

19. What’s the Necrotisingfascitiis? Necrotisingfascitiis is an uncommon soft tissue infection characterised by the progressive inflammation and necrosis of soft tissue that spread to subcutaneous tissue and fascia, causing secondary necrosis of the overlying skin and muscle. Usually this pathology is associated with septic shock. Medical illustration of advanced necrotizing fasciitis with necrosis of skin, vascular thrombosis and involvement of underlying muscle. Medical illustration depicting normal epidermal, dermal and subcutaneous anatomy.

20. Lab diagnosis • Necrotisingfascitiis should be suspected when limb pain or systemic illness is ‘disproportionate’ to the skin changes. • Possible approaches: • Computerised tomography and magnetic resonance image • The SeptiFastsystem (Roche, USA) applies the Real-time PCR technologyto molecular diagnosisof sepsi, revealingGramnegative, Grampositive and fungi, responsibles fo sepsi. Thismethod can reveal the DNA presence (startingfromanamountof 3 genomicalcopies) of the etiologicalagentresponsiblefor sepsi in lesstha 6 hour, startingfrom a 3ml blood sample. • Once necrotisingfascitiis is suspected, cultures of blood, stool and blister fluid, as well as wound specimens should be taken and aggressive management begun immediately.

22. Infection management • Supportive care and immediate administration of antibiotics improve the chance of survival. • Antibiotic recommendation include: • Doxycycline; • Third generation of cefalosporine (e.g. Ceftazidime) • A second agent in combination should be a fluoroquinolone such as ciprofloxacin or tetracycline. • The clinical course is one of systemic inflammatory response that rapidly progresses to septic shock, multiple organ dysfunction syndrome, and early death are in a high proportion on cases. Because of this rapid progression, early and aggressive debridement of wounds and amputation, are essential components of therapy and may be life saving.

25. Conclusions V. Vulnificusis a complex microorganism on many levels. While considerable progress has been made deciphering the mechanism responsible for virulence, recent studies indicate this bacterium requires a coordinated regulation of virulence factor expression in order for pathogenesis to occur. Furthermore, it appears many putative factors serve primarily in environmental survival and pathogenesis in auxiliary function. Therefore, knowledge of what factors make this bacterium successful within its natural habitat may lead to a better understanding of the virulence of this significant human pathogen.

26. Bibliography Severe necrotising fasciitis of the extremities caused by Vibrionaceae: experience of a Hong Kong tertiary hospital GM Joynt, CD Gomersall, DJ Lyon Vibriovulnificus and V. parahaemolyticus necrotising fasciitis in fishermen visiting an estuarine tropical northern Australian location. Anna Ralph a,*, Bart J. Currie b,1 Accepted 27 June 2006 DIAGNOSING AND TREATING DEADLY VIBRIO VULNIFICUS INFECTION Author: Tori L. Stivers, BSA, MS, Peachtree City, Ga Section Editor: ReneéSemonin-Holleran, RN, PhD, CEN, CCRN, CFRN, CTRN, FAEN Vibriovulnificus: Disease and Pathogenesis Melissa K. Jones and James D. Oliver* Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina 28223 Cutaneous Injury and Vibriovulnificus Infection. P.H. Chung,* S.K. Chuang,* Thomas Tsang,* Lai Wai-man,† Raymond Yung,‡ and Janice Lo‡ for the Collaborative Study Group on Vibriovulnificus Infection in Hong Kong. Necrotizing Fasciitis Caused by VibrioVulnificus in an Immunocompetent Cook Chien-Ming Chao1,2,3, Ching-Cheng Hou2, Wei-Ting Lin4,5,6