Clostridium perfringens is a Gram-positive, bacillus (rod-shaped), anaerobic, spore-forming pathogenic bacterium of the genus Clostridium. C. perfringens is present in nature and can be found as a normal component of decaying vegetation, marine sediment, the intestinal tract of humans and other vertebrates, insects, and soil. It has the shortest reported generation time of any organism at 6.3 minutes in thioglycolate medium. C. perfringens was discovered in 1891 by William H. Welch. It was originally known as Bacillus aerogenes capsulatus, followed by Bacillus welchii, before the final establishment of its current name.
Clostridium perfringens is one of the most common causes of food poisoning in the United States, alongside norovirus, Salmonella, Campylobacter, and Staphylococcus aureus. However, it can sometimes be ingested and cause no harm.
Infections induced by C. perfringens are associated with tissue necrosis, bacteremia, emphysematous cholecystitis, and gas gangrene, which is also known as clostridial myonecrosis. The specific name, perfringens, is derived from the Latin (meaning "through") and ("burst"), referring to the disruption of tissue that occurs during gas gangrene. Gas gangrene is caused by alpha toxin, or α-toxin, that embeds itself into the plasma membrane of cells and disrupts normal cellular function by altering membrane structure. Research suggests that C. perfringens is capable of engaging in polymicrobial anaerobic infections. It is commonly encountered in infections as a component of the normal flora. In this case, its role in disease is minor.
C. perfringens toxins are a result of horizontal gene transfer of a neighboring cell's plasmids. Shifts in genomic make-up are common for this species of bacterium and contribute to novel pathogenesis. Major toxins are expressed differently in certain populations of C. perfringens; these populations are organized into strains based on their expressed toxins. This especially impacts the food industry, as controlling this microbe is important for preventing foodborne illness. Novel findings in C. perfringens hyper-motility, which was provisionally thought as non-motile, have been discovered as well. Findings in metabolic processes reveal more information concerning C. perfringens pathogenic nature.
Genome Clostridium perfringens genome is between 2.9 and 4.1 million base pairs. The information on the genome of C. perfringens is still limited as there is much genetic diversity within this pathogen. It is one of the most variable gram-positive bacteria containing only 12.6 percent core genes that make up the pathogen. Despite the bacteria's diversity across strains, C. perfringens's 16S rRNA regions were found to be highly conserved (i.e. nearly identical) across all strains.
The toxin causing food poisoning in Clostridium perfringens is the CPE (Clostridium perfringens enterotoxin) cpe gene. Not all Clostridium perfringens types have the cpe gene, but all types could include this gene. The cpe gene is located in the plasmid- mediated cpe (p-cpe), or on the chromosomal cpe (c-cpe) strains. The differences in location of the cpe gene give insight into what type of infections could arise. Chromosomal cpe (c-cpe) strains are exclusively isolated from food samples, while p-cpe strains are found in non-food borne samples. c-cpe strains also produce heat resistant spores, while p-cpe strains produce heat sensitive spores. The enterotoxin–producing strain of Clostridium perfringens has been identified to be a small portion of the overall C. perfringens population (~1-5%). Plasmid DNA has been shown to play an integral role in cell pathogenesis and encodes for major toxins, including Clostridium perfringens enterotoxin.
Clostridium perfringens uses horizontal gene transfer. Through the usage of conjugation, C. perfringens can spread antibiotic resistant genes via plasmids. The pCW3 plasmid is the primary conjugation plasmid responsible for creating antibiotic resistance in C. perfringens. Furthermore, the pCW3 plasmid also encodes for multiple toxins found in pathogenic strains of C. perfringens. Antibiotic resistance genes observed thus far include tetracycline resistance, efflux protein, and aminoglycoside resistance.
Within industrial contexts, such as food production, sequencing genomes for pathogenic strains of C. perfringens has become an expanding field of research. Poultry production is impacted directly from this trend as antibiotic-resistant strains of C. perfringens are becoming more common.
Motility Clostridium perfringens is provisionally identified as non-motile. They lack flagella; however, recent research suggests gliding as a form of motility in some cultures. These hyper-motile variations of the bacteria form due to slight nucleotide polymorphisms and are regulated by various systems such as the CpAL/VirSR system and catabolite control protein A. In combination, these regulators help the spread of C. perfringens by increasing motility in regions of low nutrient densities and increasing toxicity as movement occurs.
Hyper-motile variations
In agar plate cultures bacteria with hypermotile variations like SM101 frequently appear around the borders of the colonies. Video imaging of these variants show the formation of long thin filaments that enable motion. The causes of this hypermotile phenotype and its immediate descendants were found using genome sequencing. The hypermotile offspring of strains SM101 and SM102, SM124 and SM127, respectively, had 10 and 6 nucleotide polymorphisms (SNPs) in comparison to their parent strains. The hypermotile strains also display a common trait gene mutations related to cell division.
Regulation of gliding motility The toxins that are required for some diseases caused by C. perfringens, such as myonecrosis, are regulated by the C. perfringens Agr-like (CpAl) system through the VirSR two-component system. This CpAL/VirSR system acts as a quorum sensing system encoded by other pathogenic clostridia. Studies have demonstrated that this CpAL/VirSR system also regulates C. perfringens gliding motility. This multifunctionality of the CpAL/VirSR system represents a relationship in which motility is positively and directly related to an increasing toxin production, worsening infection strength as the pathogen spreads.
Studies have also demonstrated a correlation between glucose levels and motility rates of Clostridium perfringens. As glucose levels rise, through the repression of pilT and pilD, genes that help type IV pili formation, through catabolite control protein A, gliding motility was observed to decrease. This relationship allows for C.perfringens to help spread from areas of low sugar concentration, such as the human gut, into other regions worsening infections.
Transformation There are two methods of genetic manipulation via experimentation that have been shown to cause genetic transformation in C. perfringens, the first is protoplast transformation and second is electroporation. The first method involves the variations of C. perfringens with compromised cell wall while the second uses electricity and vegetative versions of C. perfringens.
Protoplast transformation The first report of transformation in C. perfringens involved polyethylene glycol-mediated transformation of protoplasts, bacteria whose cell walls have been removed. This transformation procedure involved the addition of plasmid DNA to the protoplasts in the presence of high concentrations of polyethylene glycol. Initial protoplast transformation experiments utilized, L-phase variants, cell wall deficient versions, of C. perfringens via penicillin treatment in the presence 0.4m sucrose. However, after the transformation procedure was completed, all of the transformed cells were stuck in the form of L-phase variants, unable to regenerate cell wall. In contrast, it was observed that autoplasts, protoplasts derived from autolysis (the natural degradation of bacterial cell walls through the use of the bacteria's own enzymes), were able to regenerate and produce rods with cell walls as well as be transformed with C. perfringens plasmid DNA.
Electroporation Electroporation involves the application of a high-voltage electric field to vegetative bacterial cells for a very short period of time. This technique resulted in major advances in genetic transformation of C. perfringens, due to the bacterium often displaying itself as a vegetative cell or as dormant spores in food. The electric pulse creates pores in the bacterial cell membrane and allows the passive influx of DNA molecules. This method was found to be more efficient than the previously stated protoplast transformation due to the simpler procedure and greater yields.
Metabolic processes C. perfringens is an aerotolerant anaerobe bacterium that lives in a variety of environments including soil and human intestinal tract. C. perfringens is incapable of synthesizing multiple amino acids due to the lack of genes required for biosynthesis. Instead, the bacterium produces enzymes and toxins to break down host cells and import nutrients from the degrading cell.
C. perfringens has a complete set of enzymes for glycolysis and glycogen metabolism. In the fermentation pathway, pyruvate is converted into acetyl-CoA by pyruvate-ferredoxin oxidoreductase, producing carbon dioxide gas and reduced ferredoxin. Electrons from the reduced ferredoxin are transferred to protons by hydrogenase, resulting in the formation of hydrogen molecules () that are released from the cell along with . Pyruvate is also converted to lactate by lactate dehydrogenase, whereas acetyl-CoA is converted into ethanol, acetate, and butyrate through various enzymatic reactions, completing the anaerobic glycolysis that serves as a potential main energy source for C. perfringens. C. perfringens utilizes a variety of sugars such as fructose, galactose, glycogen, lactose, maltose, mannose, raffinose, starch, and sucrose, and various genes for glycolytic enzymes. The amino acids of these various enzymes and sugar molecules are converted to propionate through propionyl-CoA, which results in energy production.
Virulence Membrane-damaging enzymes, pore-forming toxins, intracellular toxins, and hydrolytic enzymes are the functional categories into which C. perfringens' virulence factors may be divided. These virulence factor-encoding genes can be found on chromosomes and large plasmids.
Carbohydrate-active enzymes The human gastrointestinal tract is lined with intestinal mucosa that secrete mucus and act as a defense mechanism against pathogens, toxins, and harmful substances. Mucus is made up of mucins containing several O-linked glycan glycoproteins that recognizes and forms a barrier around microbes, preventing them from attaching to endothelial cells and infecting them. C. perfringens can secrete different carbohydrate-active enzymes (CAZymes) that aid in degrading mucins and other O-glycans within the intestinal mucosa. These enzymes include: Sialidases, Hexosaminidases, Galactosidases, and Fucosidases belonging to various glycoside hydrolase families.
Sialidase Sialidases, also called neuraminidases, function to breakdown mucin by hydrolyzing the terminal sialic acid residues located within the protein through the process of desialylation. C. perfringens has three sialidases belonging to glycoside hydrolase family 33 (GH33): NanH, NanI, and NanJ. These three sialidases are typically encoded by all strains of C. perfringens.