Clostridium perfringens: Interest in human and animal pathology – Culture; Molecular diagnosis (PCR); Identification (PCR and sequencing); Molecular diagnosis of enterotoxin, phospholipase C, beta, epsilon and iota toxins codifying genes (PCR).
Information 25-03-2018.
The genus Clostridium is formed by an heterogeneous group of anaerobic sporulated gram-positive bacilli. At present, more than 200 species have been described, although only about 20 have been associated with human infection, with Clostridium perfringens being the most frequent species. Clostridium perfringens is one of the bacterial pathogens most widely distributed in the environment thanks to its ability to form spores. It can be isolated from water and soil samples, and is commonly found as part of the intestinal microflora of humans and animals. However, Clostridium perfringens can act as an opportunistic pathogen and cause infections of exogenous and endogenous origin. C. perfringens is associated with different human diseases, such as food poisoning, necrotising enteritis, gas gangrene, clostridial cellulitis and various nonspecific infections. In animals, C. perfringens causes various gastrointestinal diseases that cause significant economic losses to the livestock industry worldwide.
C. perfringens is a capsulated bacillus, immobile and capable of forming spores. This microorganism can produce powerful exotoxins responsible for causing severe toxic symptoms. The species has been divided into five types, depending on the production of the four main toxins: Clostridium perfringens type A (alpha toxin), type B (alpha, beta and epsilon toxin), type C (alpha and beta toxin), type D (alpha and epsilon toxin), and type E (alpha and iota toxin). However, along with major toxins, this microorganism can produce up to 16 toxins in various combinations, including toxins such as perfringolysin O (PFO), enterotoxin (CPE), and beta2 toxin (CPB2). These toxins are the main virulence factors of the different strains of C. perfringens. Thus, the alpha toxin (phospholipase C, lecithinase) plays a major role in the pathogenesis of gas gangrene, while beta toxin of C. perfringens type C (necrotizing and thermolabile) is involved in necrotic enteritis. On the other hand, the epsilon toxin (which promotes the generation of edema) is considered the main virulence factor of types B and D, and the iota toxin (dermonecrotic, cytotoxic and enterotoxic) mediates the infection of type E strains. Among the minor toxins, the CPE enterotoxin, is the main responsible for food poisoning caused by C. perfringens type A.
In humans, the disease is usually caused by strains of C. perfringens type A, typically associated with uncomplicated food poisoning and gas gangrene, and type C strains, associated with necrotizing enteritis. In addition, the five toxotypes can be isolated from infections in skin and soft tissues. Strains of C. perfringens type A can produce food toxicity when a large number of microorganisms are ingested (above 108), due to the release of endotoxins in the intestine. In healthy people it produces a mild and short-lasting illness, mainly causing diarrhea and abdominal pain. Toxiinfection by C. perfringens is recognized as one of the most common foodborne diseases in industrialized countries. In addition, type A strains that produce CPE are also associated with non-alimentary human intestinal diseases, including diarrhea associated with antibiotics and sporadic diarrhea. Strains of C. perfringens type C can also cause toxoinfection in humans, but are mainly associated with necrotizing enteritis. Necrotic enteritis caused by C. perfringens is rare. This disease begins as a result of the ingestion of a large number of C. perfringens bacteria type C producing β-toxin present in contaminated food. Necrotic enteritis is characterized by infection and patchy necrosis of the intestines as well as septicemia. The disease is associated with a high mortality rate, especially when the patient is not treated on time with antibiotherapy or the necrotized part is excised by surgery.
C. perfringens is also associated with various skin and soft tissue infections. In these cases, the development of the infection is associated with certain factors of the host, such as surgery or trauma, presence of serious diseases, vascular insufficiency and treatment with immunosuppressors or multiple antibiotics. The most characteristic and serious are gas gangrene and costridial cellulitis. However, the most usual isolation of these bacteria in the microbiology laboratory occurs from nonspecific infections of skin and soft tissues, bacteremia or other suppurative infections (urinary tract infections, peritonitis, cholecystitis, or surgical wound infections). Gas gangrene, associated with type A of C. perfringens, results from the growth of C. perfringens along with the production of alpha toxin in a wound, causing necrosis of the tissues, and the subsequent spread of the bacteria and systemic toxemia. If rapid and radical treatment is not performed, gas gangrene can be fatal within a short time. Second, clostridial cellulitis is an infection that characteristically affects subcutaneous or retroperitoneal tissues after trauma. Generally, the bacteria does not spread to healthy tissues and its prognosis is good.
In animals, C. perfringens causes numerous gastrointestinal infections in most species of mammals and poultry. These infections are generically called enterotoxemias because the toxins produced in the intestine can be absorbed into the general circulation. The five toxotypes of C. perfringens can be part of the normal flora of animals, and it is generally accepted that when the intestinal environment is altered, due to changes in diet or other factors, C. perfringens proliferates and produces large quantities of toxins that are absorbed into the general circulation, and are responsible for the different forms of enterotoxemias. However, some toxins produced by C. perfringens in the intestine act only locally. Among the various gastrointestinal diseases, the biotype A of C. perfringens has been associated with abomasitis and enteritis in ruminants, necrotic enteritis in chickens, and canine and feline diarrhea. While type B of C. perfringens, although it is also associated with hemorrhagic enteritis in goats, calves and foals, it is mainly known as the etiological agent of lamb dysentery. C. perfringens type C infections are manifested as necrotic enteritis that may be accompanied by enterotoxemia in almost all livestock species, mainly in neonatal animals. Animal diseases caused by types B and C of C. perfingens, producers of beta toxin, are often accompanied by sudden death or acute neurological signs, due to the passage of the same to the circulation. On the other hand, type D of C. perfringens is of upmost importance in the case of sheep and goats, responsible for sheep enterotoxemia and goat enterocolitis, respectively, diseases largely mediated by epsilon toxin, and characterized by sudden death or important neurological and respiratory signs. Occasionally, type D strains have also been isolated from infections in cattle and other animal species. Finally, toxotype E produces enterotoxemia in rabbits, and is an occasional cause of hemorrhagic enteritis and sudden death in calves. Type E disease has also been described sporadically in sheep and goats.
In addition to gastrointestinal diseases, C. perfringens can also cause skin diseases, subcutaneous and muscular tissues of different animal species. Type A of C. perfringens has been associated with the appearance of gas gangrene or malignant edema in sheep, cattle, goats and horses.
Tests carried out in IVAMI:
- Cultivation in anaerobiosis.
- PCR detection in the sample.
- Identification of isolate by sequencing.
- PCR detection of genes encoding enterotoxin and phospholipase in isolated strain.
Recommended sample:
- Cases of food poisoning: feces of the affected person, of ingested or suspect food (20 to 100 mL or g).
- Depending on the clinical picture, aspirates of wounds, abscesses or blisters (soft tissue infections), or 5 ml of blood extracted with EDTA (bacteremia) will be accepted.
- Stool samples (gastrointestinal diseases) or aspirates of the lesions (gas gangrene and tissue infections) will be accepted in animal samples.
- Other samples: Consult ivami@ivami.com
Note: Precautions will be followed for taking samples that may contain anaerobic bacteria.
Conservation and shipment of the sample:
- Refrigerated (preferred) for less than 2 days.
- Frozen: more than 2 days.
Delivery of rresults:
- Cultivation of enrichment in anaerobiosis, isolation in solid medium and identification by sequencing: 5 days.
- Direct detection by PCR in the sample: 24 to 48 hours.
- Detection of toxin production in isolated strain: 24 to 48 hours.
- Identification of isolate by sequencing: 3 days.
Cost of the test:
- Cultivation of enrichment and isolation in anaerobiosis in solid medium: Consult ivami@ivami.com.
- Direct detection by PCR in sample: Consult ivami@ivami.com.
- Isolated identification received by sequencing: Consult ivami@ivami.com.
- PCR detection of enterotoxin and phospholipase genes in isolated strain: Consult ivami@ivami.com.