Clostridium is a Gram positive bacillus, obligate anaerobe, capable of producing endospores. Clostridium species principally live in the soil and intestinal tract of many animals including humans.
One of the most clinically relevant Clostridium species is C.Perfringens
It can be classified into five subtypes on the basis of four major toxins produced by the organisms. C.perfringens type A produce C.perfringens enterotoxin ( CPE ) which is responsible for the most common
foodborne illness in the world (nearly a million cases of foodborne diseases each years ).
The principal food vehicle of C.perfringens type A is improperly cooked meat, that has cooled slowly after cooking or may have been inadequately reheating. Spores surviving the initial cooking germinate, than vegetative cells proliferate during slow cooling or insufficient reheating. (Fig.1) When the meat, contaminated by a large number of viable vegetative bacteria is eaten, the vegetative cells reach the small intestine and begin sporulating. After that, the sporulating cells undergo lysis and release CPE. (foto1)
Many bacteria, including C.perfringens have the ability to sense certain environmental stress and undergo a series of morphological changes to produce endospores that are highly stress resistant. When conditions become disadvantageous, the most drastic response to signals that indicate their immediate environment has deteriorated to a point where survival of vegetative cells is threatened is the production of an endospore.
As sporulation has a heavy energetic penalty, the initiation of the process kept under stringent control by an expanded two component signal transduction system called a phosphorelay (Fig.2).
The phosphorelay integrates multiple stimuli to ensure that cell sporulates only when all other survival strategies have been exhausted.
Environmental signals that trigger sporulation in Clostridium are not well understood. It was found that inorganic phosphate (Pi) is required for initiation of sporulation.
In culture (DS medium) little or no sporulation was observed; instead maximum sporulation efficiency was observed in the presence of 30-40 mM Pi. It has been suggested that, since an average of 15 to 30 mM concentration of Pi is normally present in a human intestinal lumen, C.perfringens might have evolved to sense mM concentration as a signal for sporulation.
Since CPE is not actively secreted but instead is released when the mother cell lyses at the completion of sporulation, so much intracellular CPE accumulates during sporulation that CPE containing paracrystalline inclusion bodies form in the cytoplasm of many sporulating C.perfringens cells.
Sporulation and CPE production are inextricably linked and several studies have demonstrated that CPE production is highly regulated by sporulation: sporulation genes (spo) encode sporulation specific sigma factors that are the major regulators of the sporulation process. The C.perfringens cpe gene encoding CPE is transcribed from three promoters (P1,P2 and P3) sigma factor dependent .
CPE consists of one peptide of 309 a.as with a molecular weight of 34262 Da. The activity of the enterotoxin is enhanced threefold by the treatment with trypsin. Trypsin cleaves at two sites: the trypsinized toxin then consist of 284 aas and two short peptides of 10 and 15 aas.
CPE binds to receptors on the surface of intestinal epithelial cells to form a pore and interacts with tight junction proteins, claudins. Binding is followed by insertion of the entire molecule into the cell membrane, but no internalization into the cell. A sudden change in ion fluxes occurs, affecting cellular metabolism and macromolecular synthesis. As intracellular calcium ion levels increase, morphological damage occurs, resulting in greatly altered membrane permeability and loss of cellular fluid and ions. (Fig.3)
Molecular insights into the initiation of sporulation in Gram-positive bacteria: new technologies for an old phenomenon
Clostridium perfringens sporulation and its relevance to pathogenesis
The enterotoxin is most active in the ileum. The diarrhea observed in humans infected with CPE-producing C.perfringens could result (at least partially) from impairment of small-intestine water absorption, which leads to a decreased intestinal absorptive capacity and increased stool mass.
The illness usually begins suddenly and lasts for less than 24 hours. Persons infected with bacteria usually don’t have fever or vomiting. Moreover illness do not pass from one person to another. Because C. perfringens is part of normal flora in many people, the laboratory confirmation of bacteria outbreaks quantified the number of organisms in bowels. If the number is greater than 106 per gram of stool in two or more ill people, C. perfringens is confirmed as the etiologic agent in an outbreak. Confirmation may also be attained by the demonstration of the enterotoxin in the stool by ELISA
Enteritis Necroticans
Gangrene of small intestine, without obvious vascular or mechanical cause, is an extremely rare disease. C.perfingens may produce a severe necrotizing diseases of the small bowel known as “enteritis necroticans”, which can occur sporadically (only a few cases have been identified in developed countries) or in an epidemic form (epidemy in Goroka, New Guinea, early in 1961). Enteritis necroticans is a life-threatening infectious diseases caused by C.perfringens type C , that releases a β-toxin (CPB).
CPB correspond to a protein which is secreted in late log growth phase, is thermo-labile, and highly-sensitive to proteases ( trypsin ).
The gene (cpb) encoding CPB is carried on a plasmid. Available information suggests that CPB is a pore forming toxin that oligomerizes to form channels in susceptible membranes. (Fig.4)
The resultant membrane permeability changes results in cell death.
In normal individuals the toxin is rapidly inactivated by trypsin in the small intestine. Clearly in times of starvation, trypsin levels are low and the levels of active β-toxin in the gut are therefore much higher.
The disease which is characterized by segmental necrosis of the proximal jejunum, is associated with high mortality rate if not diagnosed early and treated with antibiotics (Fig.5,6).
Gui L et Al. Fatal enteritis necroticans (pigbel) in a diabetic adult. Mod Pathol 2002, 15: 66-70
Irabor DA et Al. Ward Round an acute abdominal emergency. Malawi Med J 2010, 23: 78-90
It was first reported in northern Germany, after World War II, among previously starved children and adults who ate large meals of meats and it was called Darmbrand (meaning “fire bowels”). In 1963 Murrel and Roth5 reported a similar disease in patients from the Highlands of
Papua New Guinea . The patients were mostly children and young adults who presented with severe abdominal pain after eating huge quantities of sweet potatoes and inadequately cooked pork. C.perfringens was isolated from the meat and from the stools of patients, establishing the organism as the causative agent. This disease was called “pigbel”, a pidgin English term for abdominal pain after a pig feast. (Fig.7)
The ecology and epidemiology of the pig-bel syndrome in man in New Guinea"
Enteritis Necroticans pathogenetic hypothesis
Poor cooking hygiene, especially of meat, predispose to the proliferation C.perfringens type C that is normally present in pig intestine , and is also excreted in pig feces, thus poorly cooked pork was touted to be the main cause of pigbel in Papua New Guinea. Toxin production probably leads to focal paralysis, inflammation, hemorrhage and segmental gangrenous necrosis of the intestine, particularly the jejunum.
Nonetheless not all patients who ingest the C.perfringens contaminated meat developed the classical disease. An attractive hypothesis was suggested by Lawrence and Walker
Lawrence G et Al. Pathogenesis of enteritis necroticans in Papua New Guinea. The Lancet 1976, 1:125-126
that could explain the association of necrotizing enteritis with poor nutrition and episodic dietetic overindulgence. The low protein diet of Papua New Guinea highlanders is associated with low levels of digestive proteases (in particularly trypsin) in the intestinal lumen, which can inactivate the CPB. The proteases can be further blocked by the oral intake of trypsin inhibitors which are found in such dietary staples as sweet potatoes, soybean and peanut sauce in this population. Also children of New Guinea who developed enteritis necroticans had a higher rate of
Ascoris Lumbricoides infection of the gut than the general population: a potent trypsin inhibitor, secreted by the parasite to prevent is own digestion, prevents degradation of CPB.
Findings indicate a striking benefit from immunisation against pigbel in Papua New Guinea, and the program has been popular in the community because of the dread in which pigbel is held. Pigbel vaccine is relatively inexpensive, and enteritis necroticans occurs in parts of Sri Lanka, Thailand, and Vietnam, as well as in other countries: immunisation may help to prevent suffering and reduce mortality in other areas of high focal incidence.
Lawrence GW et Al. Impact of active immunization against enteritis necroticans in Papua New Guinea. Lancet, 36, 8724: 1165-67
Emerging features of Enteritis Necroticans
Since 1983 a review of 10 sporadic cases of enteritis necroticans, reported in developed countries, showed that four patients were deceased, thus indicating the high mortality associated with this condition. Six of the patient had a poorly control or untreated
diabetes mellitus which could be closely related to susceptibility to infectious diseases, an association with delayed gastric empting and reduced intestinal mobility. A well-recognised complication of diabetes is reduced gastric and small intestinal motility, caused by autonomic dysfunction in which there is a delay in small intestinal transit time. This reduced gastrointestinal motility is associated with overgrowth of bacteria that are normally present in the proximal small bowel. Diabetics may have impaired ability to degrade exogenous toxin because of decreased secretion of pancreatic protease. The clustering of diabetes among these cases indicates a susceptibility to enteritis necroticans that is not present in the general population. (Tab.1)
Matsuda et Al. Enteritis necroticans “pigbel” in a Japanese diabetic adult. Pathol Int, 2007, 57: 622-626
Moreover, in 1998 enteritis necroticans developed in a twelve-year old boy with poorly controlled diabetes mellitus after he consumed pig intestines
( chitterlings ). Histologic examinations of resected bowel tissue showed extensive mucosal necrosis, findings consistent with a diagnosis of enteritis necroticans. Probably the consumption of chitterlings by diabetic patients and other cronically ill persons can results in potentially life-threatening infectious complications.
Petrillo TM et Al. Enteritis necroticans (pigbel) in a diabetic child. N Engl J Med 2000, 342: 1250-3
Diabetic patients are vulnerable to this infection, and this disease may have been underdiagnosed in developed countries, especially when the disease is mild and self limiting. This mild and self-resolving cases may present several years later as the “scarred forms” often described as cryptogenetic multifocal ulcerous stenosing enteritis, diaphragm disease of the small intestine unrelated to non steroidal anti–inflammatory drugs, because these conditions also produce multiple segmental lesions similar to clostridial enteritis.
Francesca Serra
Diletta Gallone
