1. Pathogenetic mechanism
V. vulnificus is a mesophilic, halophilic, gram-negative bacterium that lives in the ocean. It belongs to the same genus as V. cholerae and V. parahaemolyticus . In 1970, ROLAND first reported gangrene and endotoxic shock of the calf caused by V. vulnificus infection . In 1979, FARMER named the organism Vibrio vulnificus. The pathogenic mechanism of V. vulnificus is of great significance to the prevention and treatment of infection by this bacterium . At present, research on pathogenic mechanisms mainly focuses on host defence, cytotoxicity, bacterial motility, adhesion-related proteins, virulence regulation, and biofilm formation.
1.1 Host defences include capsular polysaccharides, acid neutralization and iron overload in the body. Capsular polysaccharide (CPS) is produced by V. vulnificus and secreted to the outside of the cell, covering the external immunogenic structure of the bacteria, not only shielding it against the opsonin function of the complement system and the phagocytosis of macrophages but also helping the bacteria escape inherent immune surveillance, which can also determine the morphology of bacterial colonies and regulate the formation of biofilms . Acid neutralization means that V. vulnificus can resist gastric acid by increasing the expression of lysine decarboxylase and the expression of manganese superoxide dismutase in a low pH environment in the stomach to achieve acid neutralization . V. vulnificus obtains iron in the blood through various iron uptake systems and then grows and proliferates. Serum iron levels can indirectly reflect the infection of the body with V. vulnificus.
1.2 Cytotoxicity involves lipopolysaccharide (LPS), cytolysin and repeats-in-toxin A1 (RtxA1). LPS may play a role in cytotoxicity by affecting the activity of nitric oxide synthase (NOS) in the body; LPS can also play an adhesive role to promote the formation of biofilms, causing the infected body to develop endotoxin shock and sepsis . Cytolysin is an exotoxin encoded by the vvhA gene and is secreted outside of the cell. It exerts cytotoxicity that causes cell apoptosis and can destroy red blood cells to release iron into the blood . RtxA1 is capable of mediating cell death and tissue necrosis after contact with bacterial cells, thereby increasing the expression of the RtxA1 gene and inducing cytotoxicity and the destruction of intestinal epithelial cells and intestinal microvilli .
1.3 Bacterial motility involves flagella and fimbriae. V. vulnificus has one flagellum, which is encoded by 6 flagellum genes (flaA, flaB, flaC, flaD, flaE, flaF), of which flaB, flaD and flaC play a major role . Flagella are related to the motility, adhesion, cytotoxicity, biofilm formation and invasion of bacteria . V. vulnificus fimbriae were first discovered by GANDER under an electron microscope in 1989 . The fimbriae are related to bacterial invasion, adhesion and pathogenicity.
1.4 Extracellular proteases and adhesion-related proteins: Outer membrane protein U (OmpU) can bind to fibronectin, the main component of the mammalian extracellular matrix and is related to the adhesion function of bacteria . Membrane-bound lipoprotein A (IlpA) plays a role in adhesion and immunogenicity. The extracellular protease (ECPase) of V. vulnificus can yield a variety of ECPases that exert cytotoxic effects, including metalloprotease (Mpase), chondroitinase (ChSase), and hyaluronidase (HAase) .
1.5 Toxicity regulation includes quorum sensing (QS), total virulence regulators and haemolysin U. Bacteria can synthesize and release a kind of autoinducer (AI) that regulates many of their biological behaviours, thus causing group-sensing effects. In V. vulnificus, the synthesis of CPS and fimbriae is regulated by the QS system . The global virulence regulator cAMP-cAMP receptor protein (CRP) can bind to DNA and affect gene expression, including cytolysin, metalloproteinase and iron uptake systems, which are all regulated by the CRP system. Another virulence regulator, AphB, has a wide range of functions, including acid neutralization, motility, adhesion and pathogenicity . Haemolysin U (HlyU) is the main regulator of V. vulnificus toxicity and can regulate the expression of the rtxA1, vvhA and vvpE genes.
1.6 Biofilm formation. The biomembrane composed of CPS, exopolysaccharides (EPS) and LPS can counter the effects of drugs and the host immune system .
V. vulnificus exists as a free-living bacterium inhabiting estuarine or marine environments. Traditionally, three biotypes have been recognized: biotype 1, which accounts for almost all human infections; biotype 2, which consists primarily of eel pathogens; and biotype 3, an apparent hybrid of biotypes 1 and 2 that has been described in tilapia-associated wound infections associated with aquaculture in Israel . Wound infections most often occur in the setting of handling seafood and generally result from exposure of a wound to salt or brackish water containing the organism . Individuals with the following conditions are at increased risk for serious infection with V. vulnificus: Alcoholic cirrhosis, Underlying liver disease including cirrhosis and chronic hepatitis, Alcohol abuse without documented liver disease, Hereditary haemochromatosis, Chronic diseases such as diabetes mellitus, rheumatoid arthritis, thalassemia major, chronic renal failure, and lymphoma. Interestingly, men, and particularly older men, appear to be at much greater risk for serious infection than women . Our motherland has a vast population and a large group of patients with liver cirrhosis. The eastern coast of China has a coastline of 32,000 kilometres and a sea area of nearly 3 million square kilometres. The development of fishery operations and military activities in coastal areas makes this region a high-risk area for V. vulnificus infection.
3. Clinical characteristics
The trend of V. vulnificus infection increases with climate warming, ocean activities, and the presence of high-risk factors such as alcohol, liver disease, systemic disease, and diabetes . Traumatic bacterial infection is mainly manifested in the following clinical subtypes: traumatic infection, primary sepsis, gastroenteritis and typical clinical features. Injuries with mild symptoms may result from infection with V. vulnificus, which may cause cellulitis with mild clinical symptoms . However, in high-risk individuals (such as the female patient reported in this article) who have a history of rheumatic disease, the infection may spread quickly, causing severe myositis and necrotizing fasciitis (Fig. 1). This pathogen enters the gastrointestinal tract through contact with open wounds, thereby entering the blood system and then causing sepsis. The main disease manifestations include acute fever, chills, shock, and skin and muscle damage. People with gastrointestinal infections exhibit symptoms of acute gastroenteritis, including diarrhoea, nausea, vomiting, abdominal pain, and difficulty breathing . Typical skin and muscle damage includes local or flaky erythema and ecchymosis, bloody vesicles with exudation, necrosis and cellulitis, necrotizing fasciitis, and muscle inflammation . In the case of body infection caused by contact with seawater, skin and muscle damage rapidly progresses to necrosis and leads to severe sepsis, which may lead to life-threatening conditions . Patients usually have underlying liver disease, alcoholism, hereditary pigmentation diseases, or the chronic diseases mentioned above, which can cause primary sepsis. Approximately one-third of patients with primary sepsis will have shock or low blood pressure within 12 hours after going to the hospital.
Three-quarters of patients exhibit unique blister lesions (Fig. 2). The infection causes complications such as reduced blood circulation, diffuse intravascular coagulation, and disorders of the digestive tract. Primary V. vulnificus sepsis is an extremely serious disease with a high mortality rate.
The pathogenic diagnosis of the two patients reported in this article relied on the results of NGS. With its high-output and high-resolution characteristics, NGS not only provides us with rich genetic information but also greatly shortens the cost and time of sequencing and provides more accurate clinical guidance for the diagnosis of clinical diseases. The US Centers for Disease Control (CDC) recommended three generations of cephalosporins combined with tetracyclines as the recommended treatment for V. vulnificus infection. Patients with mild to moderate infections are usually treated with antibacterial drugs for 5–7 days. If patients with mild infections have no serious underlying diseases, mild wound infections usually respond well to topical treatment and intravenous infusion of antibacterial drugs (such as cephalosporins combined with tetracyclines or cephalosporins combined with quinolones). The length of treatment depends on the severity of the initial infection and the clinical response. Treatment of high-risk patients with severe infection: Patients who are presumed to be diagnosed with V. vulnificus sepsis should start antibacterial drugs and incision debridement as soon as possible, and when necessary, amputation plays a significant role in improving the prognosis of patients with V. vulnificus infection . For patients with severe haemodynamic instability, a low platelet count and severe coagulopathy, emergency incision and drainage should be the first choice . During the operation, the skin of the infected limb should be incised, the subcutaneous tissue should be bluntly separated, and the fascia should be exposed until the muscle membrane is formed under local anaesthesia or intravenous pain relief. The length and direction of the incision are determined according to the patient's coagulation function, skin tension, and degree of disease. After the operation, gauze soaked in iodophor and sulfamethazine solution is used for external application to facilitate dressing changes and timely assessment of the wound surface. Active treatment in the intensive care unit where possible, along with early CRRT, can help MODS patients by removing endotoxin and inflammatory mediators. Patients with acute lung injury or acute respiratory distress syndrome (ARDS) should be given non-invasive or invasive mechanical ventilation to minimize the possible consequences of low pressure and septic shock and to reduce the risk of multiple organ system failure. The affected limb should be amputated to save a patient’s life if the muscle necrosis is too severe to mend.