Clostridium perfringens- An Overview

Clostridium perfringens is a bacterium that belongs to the genus Clostridium, which consists of gram-positive, rod-shaped, anaerobic, spore-forming bacteria . Clostridium perfringens was formerly known as C. welchii or Bacillus welchii. It is widely distributed in the environment and can be found in soil, water, decaying vegetation, and marine sediments . It is also a normal inhabitant of the intestinal tract of humans and animals .

Clostridium perfringens can cause various diseases in humans and animals by producing toxins and enzymes that damage tissues, blood cells, and blood vessels . The most common diseases caused by C. perfringens are food poisoning, diarrhea, and gas gangrene . Food poisoning occurs when large numbers of C. perfringens bacteria grow in food that is improperly cooked or stored and produce a toxin that causes diarrhea and abdominal cramps after ingestion . Diarrhea can also occur when C. perfringens bacteria overgrow in the intestine due to antibiotic use or other factors. Gas gangrene is a life-threatening infection of the skin and deep tissues that results from contamination of wounds with C. perfringens spores that germinate and produce toxins and gas that destroy the affected tissues .

Clostridium perfringens is classified into five types (A to E) based on the production of four major toxins: alpha, beta, epsilon, and iota . Each type is associated with different diseases and hosts. Type A is the most common type in humans and causes food poisoning, diarrhea, and gas gangrene . Type B causes enterotoxemia (a fatal disease of the intestine) in sheep and goats . Type C causes necrotic enteritis (a severe inflammation of the intestine) in pigs, horses, cattle, sheep, and humans . Type D causes enterotoxemia in sheep, goats, and cattle . Type E causes necrotic enteritis in poultry and occasionally in other animals .

Clostridium perfringens is an important pathogen that poses a significant threat to human and animal health. It can cause outbreaks of food poisoning in large groups of people who consume contaminated food. It can also cause serious infections of wounds that can lead to amputation or death. Therefore, it is essential to prevent the growth and transmission of C. perfringens by proper cooking and storage of food, adequate hygiene and sanitation, prompt treatment of wounds, and appropriate use of antibiotics.

Clostridium perfringens is a ubiquitous bacterium that can be found in various natural and artificial environments. Some of the common habitats and sources of C. perfringens are:

• Fecal specimens from humans and animals. C. perfringens is an inhabitant of the normal intestinal flora of many vertebrates, including humans, cattle, sheep, pigs, horses, dogs, cats, and poultry . The fecal shedding of C. perfringens can contaminate soil, water, and food with spores or vegetative cells.
• Decaying vegetation, marine sediments, and soil. C. perfringens can survive in these environments as spores, which are resistant to heat, dryness, and other adverse conditions . The spores can germinate under anaerobic and nutrient-rich conditions, such as in wounds or in the gastrointestinal tract.
• Beef, poultry, gravies, and dried or pre-cooked foods. These are common sources of C. perfringens food poisoning, which occurs when food is contaminated with large numbers of C. perfringens bacteria that produce enough toxin in the intestines to cause illness . The contamination can occur during slaughter, processing, storage, or preparation of food. C. perfringens can survive high temperatures and grow rapidly between 12°C and 60°C (54°F and 140°F), which are unsafe temperatures for food holding.
• Raw meat and poultry. These are potential sources of C. perfringens infections, such as gas gangrene or clostridial myonecrosis, which are caused by the invasion of C. perfringens into deep tissues after trauma or surgery . The raw meat and poultry can harbor C. perfringens spores or vegetative cells that can enter the body through wounds or surgical sites.
• Improperly sterilized (canned) foods. These are rare but possible sources of C. perfringens infections, such as botulism-like syndrome or enteritis necroticans, which are caused by the ingestion of C. perfringens spores that germinate and produce toxins in the gut . The improperly sterilized foods can allow the survival of C. perfringens spores that can resist high temperatures and pressures.

Clostridium perfringens is a bacterium that belongs to the genus Clostridium and is Gram-positive, rod-shaped, anaerobic, and spore-forming . It can be found in nature in soil, decaying vegetation, marine sediment, and the intestinal tract of humans and animals . It can produce various toxins that cause infections and diseases in humans and animals. It is resistant to unfavorable environments because of its spores and biofilms. It is non-motile and encapsulated . It has a short generation time and can use nitrate as an electron acceptor . It is classified into seven toxinotypes based on the extracellular toxins it produces. It is considered a class B bioterrorism agent by the CDC.

Clostridium perfringens has a stable G+C content around 27–28% and average genome size of 3.5 Mb. It has a single circular chromosome with 6 million base pairs and contains 10 rRNA genes and 96 tRNA genes. Its pangenome is highly diverse, with only 12.6% core genes, making it one of the most divergent Gram-positive bacteria reported. However, its 16S rRNA regions are highly conserved (sequence identity >99.1%).

Although they lack flagella, C. perfringens bacteria are able to glide across surfaces because their bodies are lined with filaments from end-to-end. They are large rectangular bacilli with rounded or truncated ends. They are pleomorphic with straight or curved rods. Their size is about 3-8 µm X 0.4-1.2 µm . They have a thick cell wall made up of peptidoglycan that protects them from osmotic pressure. They contain spores with central or sub-terminal spores but spores are rarely seen. Endospores are able to survive long periods of exposure to air and other adverse environmental conditions. Spores are wider than the bacillary body, giving bacillus a swollen appearance resembling a spindle . They are heat resistant anaerobic bacteria that can grow under micro-aerophilic conditions .

Clostridium perfringens has a single circular chromosome made up of approximately 3.6 million base pairs, with a GC content ranging from 24 to 55% . The chromosome contains 10 rRNA genes and 96 tRNA genes. C. perfringens has a relatively low GC content compared to that of the majority of Gram-positive bacteria.

C. perfringens also harbors one or more plasmids that can vary in size and number depending on the strain. Some of these plasmids carry genes encoding toxins and other virulence factors, such as the alpha-toxin, the enterotoxin, and the binary toxin BEC . These plasmids can be transferred horizontally among C. perfringens strains by conjugation mediated by the type 4 secretion system encoded by the Pcp locus.

The genome of C. perfringens is highly dynamic and prone to rearrangements, insertions, deletions, and mutations. This contributes to the genetic diversity and adaptability of this bacterium to different environments and hosts. The genome also contains various mobile genetic elements, such as transposons, insertion sequences, and phage-related sequences, that can facilitate gene exchange and evolution.

The genome sequence of C. perfringens provides insights into its pathogenicity and physiology. For example, the genome reveals that C. perfringens lacks enzymes for the tricarboxylic acid cycle or respiratory chain, but has many enzymes for anaerobic fermentation and gas production. The genome also shows that C. perfringens has a limited capacity for amino acid biosynthesis, but has many saccharolytic enzymes and transporters for acquiring nutrients from the host or environment . Furthermore, the genome contains many genes encoding toxins, enzymes, and regulators that are involved in tissue invasion, damage, and infection .

Clostridium perfringens is an anaerobic bacterium that can grow under micro-aerophilic conditions. It has complex nutritional requirements and readily grows in media containing blood or egg. It forms large, translucent, flat, and filamentous colonies with irregular edges on solid media. It can also produce gas and acid in liquid media.

Growth conditions

• pH: Clostridium perfringens can grow in a pH range of 5.5 to 8.0, with an average pH of 7.2 .
• Temperature: Clostridium perfringens can grow in a temperature range of 20°C to 50°C, with an average temperature of 37°C . Some strains have an optimum temperature of 43°C to 47°C. At 45°C, the generation time is 10 minutes.
• Oxygen: Clostridium perfringens is an anaerobic bacterium that can grow in the absence of oxygen. It is also a microaerophilic organism that can grow in low oxygen environments.
• Water activity: Clostridium perfringens has a low water activity requirement for growth. The lowest reported water activity for growth is 0.96.
• Salt tolerance: Clostridium perfringens can tolerate up to 5% salt concentration for growth.

Cultural characteristics

Clostridium perfringens can be cultured on various media, but the most commonly used are blood agar and Robertson`s cooked meat broth. Some of the cultural characteristics of Clostridium perfringens on different media are:

• Blood agar: Clostridium perfringens produces a characteristic target hemolysis on blood agar, which consists of a double zone of beta hemolysis. The inner zone is complete hemolysis and the outer zone is partial hemolysis .
• Robertson`s cooked meat broth: Clostridium perfringens produces gas and acid in this medium, which causes the meat particles to remain intact and turn pink (saccharolytic species) or black with a foul smell (proteolytic species) .
• Litmus milk: Clostridium perfringens causes stormy fermentation and acid clot in litmus milk .
• Tryptose sulfite cycloserine (TSC) agar: This is a selective medium for the isolation and enumeration of both vegetative cells and spores of Clostridium perfringens in food and clinical samples .
• Tryptone sulfite neomycin (TSN) agar or Marshal`s medium: This is another selective medium for Clostridium perfringens that produces black colonies .
• Egg yolk agar: This medium is used for the detection of lecithinase C, which is a virulence factor of Clostridium perfringens. The presence of lecithinase C causes a precipitate around the colonies on egg yolk agar .
• MacConkey agar: Clostridium perfringens produces green fluorescent colonies on this medium .

Clostridium perfringens is a gram-positive, anaerobic, spore-forming bacillus that can ferment various carbohydrates and produce gas and acid. It can also produce several enzymes and toxins that contribute to its pathogenicity. The biochemical characteristics of C. perfringens are summarized below:

Basic characteristics

• Catalase: Negative
• Oxidase: Negative
• Indole: Positive
• Motility: Negative
• Nitrate reduction: Variable
• Spore formation: Positive (rarely seen in culture)
• Capsule: Positive
• Flagella: Negative
• Hemolysis: Positive (double zone of beta-hemolysis on blood agar)
• Lecithinase: Positive (precipitate around colonies on egg yolk media)
• Shape: Rod-shaped with blunt ends
• Gram stain: Gram-positive

Fermentation of carbohydrates

C. perfringens can ferment various sugars and produce acid and gas. The fermentation patterns of some common carbohydrates are:

• Glucose: Positive
• Fructose: Positive
• Galactose: Positive
• Lactose: Positive
• Maltose: Positive
• Mannitol: Positive
• Mannose: Positive
• Sucrose: Positive
• Arabinose: Negative
• Cellobiose: Variable
• Glycerol: Weakly positive
• Glycogen: Variable
• Inositol: Variable
• Inulin: Negative
• Melibiose: Negative
• Raffinose: Variable
• Ribose: Variable
• Salicin: Negative
• Sorbitol: Negative
• Starch: Variable
• Trehalose: Variable
• Xylose: Negative

Enzymatic reactions

C. perfringens can produce several enzymes that hydrolyze different substrates and enhance its virulence. Some of the enzymatic reactions are:

• Amylase: Positive (hydrolyzes starch)
• Elastase: Positive (hydrolyzes elastin)
• Esculin hydrolysis: Variable (hydrolyzes esculin to esculetin and glucose)
• Gelatin hydrolysis: Positive (hydrolyzes gelatin to peptides and amino acids)
• Hyaluronidase: Positive (hydrolyzes hyaluronic acid in connective tissue)
• Lipase: Negative (does not hydrolyze fats)
• Neuraminidase: Positive (hydrolyzes sialic acid in glycoproteins and glycolipids)
• ONPG (beta-galactosidase): Positive (hydrolyzes o-nitrophenyl-beta-D-galactopyranoside to o-nitrophenol and galactose)
• Ribonuclease: Positive (hydrolyzes RNA to nucleotides)
• Superoxide dismutase: Positive (converts superoxide radicals to hydrogen peroxide and oxygen)

Clostridium perfringens is a gram-positive, anaerobic, spore-forming rod that produces a variety of toxins and enzymes that contribute to its pathogenicity. The bacterium can cause different diseases depending on the type and amount of toxins produced. C. perfringens is classified into five toxinotypes (A, B, C, D, and E) based on the production of four major toxins: alpha (CPA), beta (CPB), epsilon (ETX), and iota (ITX). These toxins are responsible for the most severe syndromes caused by C. perfringens, such as gas gangrene, enteritis necroticans, and enterotoxemia. In addition to these major toxins, C. perfringens can produce other minor toxins and enzymes that have various effects on the host tissues and cells.

The following table summarizes the main virulence factors and toxins produced by C. perfringens and their roles in disease:

Some additional sentences to conclude the point are:

C. perfringens produces a diverse array of virulence factors and toxins that enable it to cause a wide spectrum of diseases in humans and animals. The type and amount of toxins produced depend on various factors such as strain type, environmental conditions, host susceptibility, and bacterial growth phase. The toxins exert their effects by targeting different cellular components and pathways, resulting in cytotoxicity, tissue damage, inflammation, vascular dysfunction, neurological impairment, diarrhea, or death. Understanding the mechanisms of action and regulation of these toxins is essential for developing effective strategies for diagnosis, treatment, and prevention of C. perfringens infections.

Clostridium perfringens can cause various diseases in humans and animals, depending on the type of toxin produced by the bacteria. The main diseases caused by C. perfringens are:

• Gas gangrene: This is a life-threatening infection of soft tissues, especially muscles, that results from the introduction of C. perfringens spores into wounds. The spores germinate and produce toxins that destroy the tissue and cause gas production, leading to swelling, pain, foul-smelling discharge, and shock. Gas gangrene is most commonly caused by C. perfringens type A, which produces alpha toxin, a potent lecithinase that damages cell membranes and blood vessels .
• Food poisoning: This is a common illness that occurs when food contaminated with large numbers of C. perfringens bacteria or spores is consumed. The bacteria multiply in the intestines and produce enterotoxin, which causes diarrhea and abdominal cramps. Food poisoning is usually caused by C. perfringens type A, which produces CPE (Clostridium perfringens enterotoxin), a toxin that binds to intestinal epithelial cells and disrupts their tight junctions .
• Enteritis necroticans: This is a rare but severe form of food poisoning that occurs when food contaminated with C. perfringens type C is ingested. Type C produces beta toxin, a necrotizing toxin that damages the intestinal mucosa and causes hemorrhagic necrosis, perforation, and peritonitis. Enteritis necroticans is also known as pigbel, because it affects people who consume undercooked pork contaminated with pig intestines .
• Necrotic enteritis: This is a disease that affects poultry, especially chickens, and causes necrosis of the intestinal mucosa, diarrhea, and mortality. It is caused by C. perfringens type A or C, which produce alpha toxin and/or beta toxin.

• Other infections: C. perfringens can also cause other types of infections, such as cellulitis, fasciitis, septicemia, endocarditis, meningitis, and arthritis. These infections are usually associated with trauma, surgery, or immunosuppression.

  Clinical features and diagnosis of Clostridium perfringens infections

Clostridium perfringens can cause different types of infections in humans, depending on the type of toxin produced by the bacteria. The most common infections are food poisoning, gas gangrene, and necrotizing enteritis.

Food poisoning

C. perfringens food poisoning is one of the most common causes of foodborne illness in the United States. It is usually caused by type A strains that produce an enterotoxin (CPE) in the intestines after ingestion of contaminated food.

The symptoms of C. perfringens food poisoning include:

• Diarrhea
• Abdominal cramps
• Nausea
• No vomiting or fever

The symptoms usually begin 6 to 24 hours after eating the contaminated food and last for less than 24 hours. The illness is usually mild and self-limiting, but can be severe and life-threatening in some cases, especially in elderly or immunocompromised people.

The diagnosis of C. perfringens food poisoning is based on:

• The clinical presentation and history of exposure to suspect food
• The detection of CPE toxin or C. perfringens bacteria in stool samples
• The isolation and identification of C. perfringens from food samples

The laboratory tests for C. perfringens food poisoning include:

• Enzyme immunoassay (EIA) or polymerase chain reaction (PCR) for CPE toxin in stool samples
• Culture and enumeration of C. perfringens spores (>10^6 spores/g) in stool samples
• Culture and biochemical identification of C. perfringens from food samples
• Nagler`s reaction for lecithinase activity on egg yolk agar plates

Gas gangrene

Gas gangrene is a severe and potentially fatal infection of soft tissues caused by type A strains that produce alpha-toxin and other toxins. It usually occurs after traumatic injuries or surgical procedures that introduce spores into deep wounds.

The symptoms of gas gangrene include:

• Severe pain and swelling at the site of infection
• Foul-smelling discharge and gas production
• Tissue necrosis and gangrene
• Fever, hemolysis, shock, and multiorgan failure

The diagnosis of gas gangrene is based on:

• The clinical presentation and history of trauma or surgery
• The presence of large gram-positive rods with spores in Gram-stained smears from wound specimens
• The culture and identification of C. perfringens from wound specimens
• The detection of alpha-toxin or other toxins in wound specimens or serum samples

The laboratory tests for gas gangrene include:

• Gram stain and microscopy of wound specimens
• Culture and biochemical identification of C. perfringens from wound specimens
• Toxin neutralization test with specific antitoxins for alpha-toxin or other toxins
• PCR or EIA for toxin genes or proteins in wound specimens or serum samples

Necrotizing enteritis

Necrotizing enteritis is a rare but serious infection of the small intestine caused by type C strains that produce beta-toxin and other toxins. It usually affects children in developing countries who have poor nutrition and hygiene.

The symptoms of necrotizing enteritis include:

• Severe abdominal pain and distension
• Bloody diarrhea
• Vomiting
• Fever, dehydration, shock, and death

The diagnosis of necrotizing enteritis is based on:

• The clinical presentation and history of exposure to contaminated food or water
• The detection of beta-toxin or C. perfringens bacteria in stool samples
• The histopathological examination of intestinal biopsies

The laboratory tests for necrotizing enteritis include:

• EIA or PCR for beta-toxin in stool samples
• Culture and enumeration of C. perfringens (>10^5 CFU/g) in stool samples
• Culture and biochemical identification of C. perfringens from stool samples

• Histopathology and immunohistochemistry of intestinal biopsies

  Treatment and prevention of Clostridium perfringens infections

The treatment and prevention of Clostridium perfringens infections depend on the type and severity of the infection . Here are some general guidelines:

For food poisoning

• Oral rehydration is usually the only treatment required, and antibiotics are not useful .
• Drink extra fluids as long as diarrhea lasts to prevent dehydration.
• Over-the-counter oral rehydration salts are available for mild dehydration.
• Severe dehydration may require hospitalization.

For tissue infections

• Prompt and extensive surgical removal of the affected area and penicillin are the standard treatment .
• Hyperbaric oxygen may be of help in the medical management of clostridial tissue infections.
• Antitoxins and hyperimmune plasma may be needed for some cases, such as foals with enterotoxemia .

For prevention

• Cook food to a safe temperature to kill germs. Use a food thermometer to check, especially whole poultry and large meat roasts.
• Keep cooked food at 140°F or hotter or 40°F or colder if it will not be served and eaten soon.
• Refrigerate leftovers at 40°F or colder within 2 hours after cooking the food or removing it from an appliance that’s keeping it at a safe temperature. Refrigerate within 1 hour if the food is exposed to temperatures above 90°F, like a hot car or picnic.
• Divide large pots of food, such as soups and stews, and large cuts of meats, such as roasts, into small quantities to help it cool quickly in the refrigerator.
• Leftovers should be reheated to at least 165°F before serving.
• Avoid eating food that tastes, smells, or looks different from what it is supposed to.
• In case of tissue infections, early and adequate cleansing of contaminated wounds and surgical debridement, together with the administration of antimicrobial drugs directed against clostridia (eg, penicillin), are the best available preventive measures.
• Antitoxins should not be relied on. Although toxoids for active immunization have been prepared, they have not come into practical use.
• Vaccination of mares and good biosecurity and hygiene are vital for preventing enterotoxemia in foals .

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