Clostridium Perfringens BTY618

Clostridium perfringens BTY618 Clostridium perfringens (Cl.welchii) Clostridium perfringens (formerly known as C. welchii) is an anaerobic, large Gram positive spore forming bacillus. C. perfringens is ubiquitous in nature and can be found in soil, water and gastrointestinal tract (GIT) of humans and other vertebrates and insects. Morphology – Large Gram-positive bacilli with straight, parallel sides & slightly rounded ends. Measure 4-6x1μm in size , occurring singly or in chains. Pleomorphic, capsulated & non-motile. Spores are central or sub terminal. Spores are rarely seen in culture media or material from pathogenic lesions, a characteristic morphologic feature. CULTURAL CHARACTERISTICS – Robertson’s cooked meat broth is ideal; meat is turned pink but not digested with sour odour. -Stormy fermentation of lactose in litmus milk; the acid coagulates casein-acid clot. -On BAM: Target haemolysis BIOCHEMICAL REACTIONS: Glucose Indole –ve. Lactose fermented with MR +ve Maltose A & G production VP –ve. & Nitrate reduction test +ve. Toxins  Clostridium perfringens Type A Clostridium perfringens Type B Clostridium perfringens Type C Clostridium perfringens Type D Clostridium perfringens Type E Clostridium perfringens Strain of Clostridium perfringens Type A Type B Type C Type D Toxins Alpha Disease(s) Myonecrosis in human and animals/gas gangrene in human being. Alpha, beta, epsilon Severe enteritis in young calves, foals, lambs, and piglets./lamb dysentery Alpha, beta Necrotizing enterocolitis in humans/struck. Alpha, epsilon Enterotoxemia in sheep and goats and, on rare occasions, in cattle/pulpy kidney disease in lamb. Enterotoxemia in Alpha toxin (CPA). Virtually all C. perfringens isolates produce CPA, a zinc-containing phospholipase C enzyme of 370 amino acids. consists of a membrane-binding C-domain composed of a β- sheet, a catalytic N-domain composed of α-helices, and a central loop domain containing a ganglioside GM1a binding site CPA is thought to use ganglioside GM1 as a receptor. Through the hydrolysis of phosphatidylcholine and sphingomyelin in the plasma membrane, CPA induces the formation of diacylglycerol (DAG) and ceramide (CER), respectively CPA can activate endogenous host enzymes with similar phospholipase and sphingomyelinase activities In addition to these enzymatic events in the plasma membrane, binding to GM1 allows CPA to interact with the tropomyosin receptor kinase A (TrkA), leading to activation of the MEK/ERK pathway Beta toxin (CPB). C. perfringens type B and C isolates produce CPB as a pro-toxin of 336 amino acids, containing a 27-amino acid signal sequence that is cleaved during secretion. This results in a mature toxin of ~35 kDa CPB is a β-pore-forming toxin (PFT) of the α-hemolysin family and shares amino acid sequence similarity with the β-PFTs of Staphylococcus aureus CPB is an oligomerizing toxin that forms small functional pores in the plasma membrane of susceptible cells. These pores allow the entry of Ca2+, Na+ and Cl− into the cells, inducing cell swelling In porcine aortic endothelial cells, CPB induces rapid disruption of the actin cytoskeleton, cell shrinkage, and cell border retraction Efflux of potassium also occurs through these CPB pores, inducing phosphorylation of p38 MAP and JNK kinases, both of which can activate pathways associated with host cell adaptation and survival C. perfringens enterotoxin (CPE). CPE, a 35 kDa single polypeptide, is produced by all type F strains and by some type C, D, and E strains The CPE protein consists of a C-terminal receptor-binding domain and an N-terminal cytotoxicity domain that mediates oligomerization and membrane insertion during pore formation The cellular action of CPE starts with its binding to receptors, which include certain claudins Claudins are a large family of proteins important for maintaining the structure and function of tight junctions made by epithelial and endothelial cells Claudins contain two extracellular loops (ECLs), both of which participate in CPE binding Once bound to a claudin receptor, CPE becomes localized in an ~90 kDa small complex that also contains a receptor claudin and the nonreceptor claudin-1 Approximately six small complexes oligomerize to form a ∼450 kDa prepore on the plasma membrane surface Each CPE in the prepore then extends a β- hairpin loop that assembles into a β-barrel that inserts into the membrane to create a 1.4 nm pore The CPE pore is permeable to small molecules, particularly cations Epsilon toxin (ETX). ETX, produced only by type B and D isolates, has structural similarity to the aerolysin toxin produced by Aeromonas sp., so it is classified as a β-PFT of the aerolysin family While there is still some uncertainty about the ETX receptor, the Myelin and Lymphocyte (MAL) protein is emerging as a strong receptor candidate. After binding, ETX forms a heptameric prepore followed by the insertion of a β-barrel that allows efficient pore formation ETX is released as a weakly active prototoxin of ~33 kDa. Several intestinal proteases such as trypsin, α- chymotrypsin, and carboxypeptidases remove N-terminal and C-terminal residues from this protoxin, resulting in a mature, active protein that is 1000 times more toxic than the prototoxin It has also been demonstrated that ITX entrance into host cells involves cell-surface antigen CD44-associated endocytosis After endocytosis, Ia translocates from late endosomes into the cytoplasm where it exerts ADP-ribosylating activity involving the covalent attachment of ADP-ribose onto an Arg at residue 177 of actin This effect induces depolymerization of actin filaments, which increases the presence of G-actin monomers Iota toxin (ITX). ITX, a binary toxin produced only by C. perfringens type E strains, is comprised of an enzyme component (Ia) and a binding component (Ib) Removal of a ~ 20 kDa N-terminal fragment by trypsin or chymotrypsin is required to produce an active Ib, which is initially synthesized as an inactive toxin of ~100 kDa These proteases also cleave off small peptides (9 to 13 amino acid residues) from the N-terminus of the Ia precursor, producing an active form The lipolysis-stimulated lipoprotein (LSR, also known as angulin-1) is a cellular receptor for Ib This toxin component binds to the LSR N-terminal 10 to 15 residues, followed by endocytosis of ITX (together with LSR) into trafficking endosomes has also been demonstrated that ITX entrance into host cells involves cell-surface antigen CD44-associated endocytosis Neuraminidase: Neuraminidase is a key enzyme for the catabolism of sialic acidcontaining oligosaccharides. Red blood cells (RBCs) have a net negative surface charge due to ionized sialic acid. Neuraminidase makes RBCs pan agglutinable removing sialic acid, resulting in an increase in blood viscosity and promoting capillary thrombosis. Enterotoxin: Enterotoxin is the major toxin responsible for human food poisoning. Clostridium perfringens enterotoxin (CPE) is responsible for causing the symptoms of C. perfringens type A food poisoning. Sources of infection can be either: Exogenous: injury, contamination of wound by animal faeces. Endogenous: from own GIT after disruption of mucous membrane, often due to a mixed infection which can enhances an anaerobic environment, thereby encouraging the organism to grow. Infections caused by C. perfringens can lead to: Skin/Wound infections: superficial skin infection, anaerobic cellulitis, myonecrosis or gas gangrene and puerperal infections. A pus swab is required to confirm the cause of infection. Septicemia (can lead to Disseminated Intravascular Coagulation). A Blood culture specimen will be required to confirm this. Self-limiting food poisoning (watery diarrhea) where enterotoxin was produced in the meat. Incubation period is 6 to 24 hours after ingestion. If large dose is ingested it can lead to Necrotizing Enteritis. A Stool specimen is required to confirm the diagnosis. Pathogenesis of Clostridium perfringens Invasive infection and Gas gangrenes In invasive clostridial infections, spores reach tissue either by contamination of traumatized areas (soil, feces) or from the intestinal tract. The spores germinate at low oxidation-reduction potential; vegetative cells multiply, ferment carbohydrates present in the tissue, and produce gas. The distention of tissue and interference with blood supply, together with the secretion of necrotizing toxin and hyaluronidase, favor the spread of infection. Tissue necrosis extends, providing an opportunity for increased bacterial growth; hemolytic anemia; and, ultimately, severe toxemia and death. The toxin involved in gas gangrene is known as α-toxin, which inserts into the plasma membrane of cells, producing gaps in the membrane that disrupt normal cellular function. Clinical Features of Clostridium perfringens Gas gangrene Clostridium perfringens is the most common strain of clostridia associated with trauma-induced gas gangrene in humans and is also a major cause of spontaneous (nontraumatic) gas gangrene. The classic features of gas gangrene are extensive local tissue destruction progressing to profound shock and death. From a contaminated wound (eg, a compound fracture, postpartum uterus), the infection spreads in 1–3 days to produce crepitation in the subcutaneous tissue and muscle, foul-smelling discharge, rapidly progressing necrosis, fever, hemolysis, toxemia, shock, and death. At times, the infection results only in anaerobic fasciitis or cellulitis. Food Poisoning C.perfringens food poisoning usually follows the ingestion of large numbers of clostridia that have grown in warmed meat dishes. The toxin forms when the organisms sporulate in the gut, with the onset of diarrhea and abdominal cramps—usually without vomiting or fever—in 7–30 hours. The illness suddenly begins and lasts only 1–2 days. Nagler’s Test C. perfringens can be diagnosed by Nagler’s reaction, where the suspect organism is cultured on an egg yolk media plate. One side of the plate contains anti-alpha-toxin, while the other side does not. A streak of a suspect organism is placed through both sides. An area of turbidity will form around the side that does not have the anti-alpha-toxin, indicating uninhibited lecithinase activity. Prevention of Clostridium perfringens Infection The growth of C. perfringens spores can be prevented by most importantly cooking food, especially beef, and poultry, thoroughly, to the recommended temperatures. Leftover food should be refrigerated to a temperature below 4 °C within two hours of preparation. Large pots of food such as soup or stew with meats should be divided into small quantities and covered for refrigeration. Leftovers should be reheated to at least 74 °C before serving. A rule of thumb is that if the food tastes, smells, or looks different from what it is supposed to, then the food should be avoided. Even if it looks safe, food that has been out for a long time can also be dangerous to eat. 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. Clostridium chauvoei  Clostridium chauvoei is an anaerobic, motile, Gram-positive bacterium.  It is a soil-borne pathogen that can cause blackleg in cattle and sheep.  In cattle affects young stage (6 months to 2years ) during rainy season.  Affect specially in hind and fore quarters of animals.  In sheep due to wound, lambing docking, shearing and castration. Disease –Blackleg/blackleg baillus /Black quarter (B.Q.) in cattle and sheep. Symptoms of Blackleg in cattle Depression, anorexia, rumen stasis, high fever (41-42 degrees), and tachycardia. These signs are usually followed by marked lameness with noticeable muscle swelling of the affected leg’s upper part. Edematous and crepitate swelling develops in the hip, shoulder, chest, back, neck, or elsewhere. The swelling is small, hot, and painful, and then swelling enlarges, and there is crepitation on palpation. The skin is often discolored in affected areas, and on the cut section, a serosanguineous rancid fluid appears and gets escapes. How to Diagnosis the Black Quarter in Cattle? A rapidly fatal, febrile disease in well-nourished young cattle with the massive muscles’ crepitant swellings suggests Blackleg. The affected muscle is a dark red to black, dry and spongy, it has a sweetish odor and is filtered with small bubbles, but with edema, suggests Blackleg. Field diagnosis can be made on history, clinical findings and necropsy examination, history of vaccination, history of recent transferring of the vicinity soil inside and outside the barn, course of the disease is per acute and acute, and outbreak six months to 2 years of age in