Capnocytophaga canimorsus

History

Capnocytophaga canimorsus was first observed in 1976 by Bobo and Newton. The pair isolated a previously unknown Gram-negative bacteria from a patient presenting with meningitis in addition to septicemia. The patient had been previously exposed to two canine bites on two consecutive days from two different dogs. Noting the coincidence between the timing of the bites with the onset of symptoms, Butler et al. analyzed 17 similar cases of patients presenting with either septicemia or meningitis from 1961-1975. The cases had been sent to the CDC for examination due to the presence of an unknown Gram-negative bacillus isolated from infected individuals. Butler notified the CDC of the high incidence of dog bites in connection with the infections. The CDC could not identify the organism and so they applied the name CDC group DF-2. DF-2 stands for Dysgonic fermenter, meaning that the bacteria is a slow-growing, fermentative bacillus. In 1989, while analyzing the properties of the unknown bacteria, Weaver et al. noted many similarities to bacteria of the genus Capnocytophaga. Later that same year, Brenner et al. proposed the name Capnocytophaga canimorsus after examining the morphology, G+C% content and motility of the bacteria.

Epidemiology

In the United States, 50% of Americans will be bitten by dogs during the course of their lifetime; 1 million Americans are bitten by dogs annually. Cases of human infection following exposure to C. canimorsus have been observed worldwide. Cases have been reported in the United States, Canada, Europe, Australia and S. Africa. Symptoms may appear within 2–3 days post-exposure, or up to four weeks after. Middle-aged and elderly persons are at greater risk for contraction of disease; more than 60% of sufferers are fifty years of age or older. In addition, individuals who spend a greater portion of their time with canines and felines are also in a higher risk category. This includes veterinarians, breeders, pet owners, and keepers. Having certain pre-existing medical conditions exacerbates the risk. Chance of infection after dog bites varies between three and twenty percent; for cats, it may be as high as 50%.

Morphology, culture and isolation

C. canimorsus is a fastidious, Gram-negative, fermentative, non spore-forming rod. Bacilli are usually 1-3 μm in length. After growth on agar plates, longer rods tend to have a curved shape. The bacteria does not have flagella but rather moves with a gliding motion, although this can be difficult to see. C. canimorsus requires the right medium for growth. The bacteria cultures well on blood agar plates (heart infusion agar with 5% sheep or rabbit blood) and chocolate agar plates. Colonies may not be visible for up to 48 hours due to slow growth. At 18 hours, colonies are usually less than 0.5 mm. in diameter, and are spotty and convex. At 24 hours, colonies may be up to 1 mm. in diameter. After 48 hours, colonies are narrow, flat and smooth, with spreading edges. At this time, colonies may appear to be purple, pink, or yellow, but once they are scraped from the agar plate they are always yellow in appearance.

Genome

The genome of Capnocytophaga canimorsus strain Cc5 consists of a single circular chromosome of 2,571,406 bp with a G+C content of 36.11%, and it encodes 2,405 open reading frames (ORFs). The Cc5 genome contains 46 tRNAs, three sets of rRNA, an RNase P, two tmRNAs, a TPP riboswitch, and an SRP, and it contains one CRISPR region. It does not encode any type III, IV, or VI secretion system, which are commonly linked to pathogenesis. The annotated genome sequence of Cc5 was deposited in GenBank under accession number CP002113.

Capnocytophaga canimorsus in animals

C. canimorsus belongs to the genus Capnocytophaga. Members of this genus are found in the oral cavities of humans and animals. A majority of these species are not found in humans. Capnocytophaga canimorsus is a commensal bacteria found in dogs and cats; it is not a member of the normal microbiology of humans. Approximately 26% of dogs carry the commensal bacteria in their mouths. C. canimorsus rarely causes disease-like symptoms in animals. There has been one reported case of C. canimorsus isolated from a dog bite wound on a small dog's head; the bacteria was localized to the wound and the dog did not present with bacteremia. There have been a few cases of infection reported in rabbits after being bitten by dogs. Clinical manifestations of C. canimorsus in rabbits causes a range of symptoms, including Disseminated Intravascular Coagulation (DIC), cellular necrosis (tissue death), low blood pressure, gangrene, and kidney failure.

High-risk categories

In addition to those at higher risk of developing complications from C. canimorsus due to greater contact with felines and canines, certain pre-existing conditions place individuals in a critically high-risk category. Among these are those who have undergone a splenectomy, alcoholics, and individuals with immunosuppression due to the use of steroids such as glucocorticoids. Individuals with β-Thalassemia and smokers are also listed as high-risk. These individuals, like asplenics and alcoholics, have increased levels of alimentary iron in their bloodstream. C. canimorsus requires large amounts of iron to grow, so these conditions are optimal for the bacillus.

Asplenia

Of the cases presented in literature, 33% occurred in asplenic individuals. These individuals have decreased IgM and IgG production. They also have delayed macrophage assembly and produce less tuftsin. Tuftsin is responsible for the stimulation of phagocytosis, so its decrease in the presence of bacterial infection poses a problem. A functional spleen is important for the removal of pathogens. Because this particular pathogen seems to flourish in asplenic patients, it is suggested that both IgM antibodies and tuftsin are critical in the process of marking this bacteria for destruction by phagocytosis. Asplenics are often associated with double the amount of healthy iron in the bloodstream, and are sixty times more at risk of developing fatal clinical manifestations of the bacteria. Individuals with asplenia often experience symptom onset within a day of exposure. The infection rapidly progresses toward multiple organ system failures and finally death. The mortality rate in individuals with asplenia is much higher than any other at risk-category for C. canimorsus infections.

Alcoholism

Alcoholics represent approximately 24% of individuals presenting with C. canimorsus infections. Alcoholism has been shown to result in decreased superoxide production in neutrophils as well as declines in neutrophil elastase activity. This results in an increase in predisposition to bacteremia (bacteria in the blood). As a result, people suffering from alcoholism are more likely to suffer from the more dangerous aspects of C. canimorsus invasions. Finally, alcoholics are associated with increased blood iron content.

Immunosuppression

Immunosuppresants are often used to battle autoimmune diseases such as lupus. When an individual undergoes treatment with immunosuppressants such as glucocorticoids, their body's defenses are chemically lowered. As a result, exposure to C. canimorsus is more infectious in these individuals than in healthy individuals. Immunosuppressed patients make up approximately 5% of individuals presenting with C. canimorsus symptoms.

Symptom onset and clinical manifestations

Symptoms appear within 1–8 days after exposure to C. canimorsus but usually present around day 2. Symptoms range from mild flu-like symptoms to full-blown fulminant septicemia. Individuals often complain of any combination of the following: fever, vomiting, diarrhea, malaise, abdominal pain, myalgia, confusion, dyspnea, headaches, and skin rashes such as exanthema. More severe cases of endocarditis, DIC, and meningitis have been reported. Prior treatment with methylprednisolone has been shown to prolong bacteremia in these infections, which enables the progression of endocarditis.

Differential diagnosis

Diagnosing infections with C. canimorsus can be difficult. Common practice for culturing isolates is to keep agar plates for one week; sometimes cultures of C. canimorsus are not visible at that point due to slow growth or inappropriate media. C. canimorsus requires very specific culture media and conditions; enriched media is necessary. C. canimorsus displays enhanced growth in high concentrations of carbon dioxide, therefore it is necessary to culture the bacteria in candle extinction jars or carbon dioxide incubators. To diagnose this bacillus, certain reactions may be tested. The bacteria should test positive for catalase and oxidase, arginine dihydrolase, maltose, and lactose. It should test negative for nitrate reduction, urease, and H2S production. C. canimorsus can be distinguished from other Gram-negative bacteria by testing negative for inulin and sucrose. Due to the relatively slow growth of this bacteria, diagnosis often relies upon the clinician having knowledge that the patient was previously in contact with a canine or feline. Once aware of this, clinicians can request that agar plates be kept longer than one week to ensure proper isolation of the bacteria. Sometimes, even these methods fail. Cases have been noted where cultures repeatedly came up negative for C. canimorsus, only to determine its presence with 16S rRNA gene sequencing. PCR assays of species specific genes may also be beneficial. For individuals presenting with meningitis, C. canimorsus can be diagnosed with a CSF Gram stain. These methods are more costly but are the best way to ensure species-level identification. Isolates are usually obtained from blood cultures (88% of the time) and less frequently from bite wounds. In incidents where the patient is in full septic shock, whole blood smears may be effective.

Treatment

Immediate cleansing of wounds caused by canines and felines can be successful in keeping C. canimorsus infections at bay. Irrigation of wounds with saline is recommended and individuals are encouraged to seek medical help for the administration of antibiotics. Antibiotics are recommended if wounds are deep or individuals prolong seeking medical attention. Antibiotics that contain beta-lactamase inhibitors (i.e., oral Augmentin or parenteral Unasyn) cover C. canimorsus as well as other organisms common in bites.

Penicillin G is the drug of choice, although there have been some isolates found to show resistance. C. canimorsus is susceptible to ampicillin, third generation cephalosporins, tetracyclines, clindamycin, and chloramphenicol. It has shown resistance to gentamicin. Treatment is recommended for a minimum of three weeks. Hospitalization is required in more severe infections. For cases of septicemia, high doses of penicillin are required. For those with a penicillin allergy, there is no alternative that is as effective as penicillin. To control DIC, a condition often associated with sepsis, plasmaphoresis (separation and removal of blood plasma from blood cells) and/or leukapheresis (removal of excess white blood cells) are often utilized. Third generation cephalosporins are often given prior to diagnosis because they cover a broad range of Gram-negative bacteria. After diagnosis, provided the strain is not beta-lactamase producing, medication should be switched to penicilin G. Presumably penicillin G could be given with a beta-lactamase inhibitor combination, such as Unasyn, for patients with a beta-lactamase producing strain.

Mortality of meningitis-related infections is much lower than mortality associated with septicemia. Because C. canimorsus induces fulminant septicemia, the faster the diagnosis, the better the chance of survival.

Evasion of immune system

Capnocytophaga canimorsus has been observed to multiply in the presence of mouse J774.1 macrophages. Macrophages recognize and kill pathogens by engulfing them. They also secrete cytokines necessary to begin the immune pathway cascade. C. canimorsus bacteria are not internalized by macrophages; in fact, macrophage monolayers break down in their presence, suggestive of a cytotoxin. In the presence of C. canimorsus, cytokine activity is greatly downregulated, because the macrophages fail to produce TNF-α, IL-8, IL-6 and IL-1α, interferon-γ, and nitric oxide. In addition, TLR4 (Toll-like Receptor 4) normally recognizes pathogens and begins a signalling cascade to induce production of pro-inflammatory cytokines via the NF-κB pathway. In cells infected with C. canimorsus, TLR4 did not activate the signalling pathway and therefore did not elicit an inflammatory response by the immune system. Because this bacterium doesn't elicit a strong inflammatory response, the bacteria have ample time for replication before detection by the host immune system. Electron micrographs of J774.1 monolayers infected with C. canimorsus have shown cells of the bacteria within the macrophage's vacuoles, surrounded by bacterial septa. This suggests that C. canimorsus replicates intracellularly inside of macrophages. C. Canimorsus cells also show resistance to killing by complement and killing by polymorphonuclear leukocytes. C. canimorsus, when in the presence of PMNs, feeds on them by deglycosylating host glycans. In fact, in the presence of PMNs, C. canimorsus experiences robust growth. C. canimorsus has the ability to evade these necessary immune functions, and therefore it must be taken seriously. Greater knowledge about the pathogenesis of this bacillus is required in order to prevent and treat the diseases associated with it.