Lecture 22
Causative agents of anaerobe infections
Clostridia
The genus Clostridium consists of relatively large, Gram-positive, rod-shaped bacteria in the Phylum Firmicutes (Clostridia is actually a Class in the Phylum). All species form endospores and have a strictly fermentative type of metabolism. Most clostridia will not grow under aerobic conditions and vegetative cells are killed by exposure to O2, but their spores are able to survive long periods of exposure to air.
The clostridia are ancient organisms that live in virtually all of the anaerobic habitats of nature where organic compounds are present, including soils, aquatic sediments and the intestinal tracts of animals.
Clostridia are able to ferment a wide variety of organic compounds. They produce end products such as butyric acid, acetic acid, butanol and acetone, and large amounts of gas (CO2 and H2) during fermentation of sugars. A variety of foul smelling compounds are formed during the fermentation of amino acids and fatty acids. The clostridia also produce a wide variety of extracellular enzymes to degrade large biological molecules (e.g. proteins, lipids, collagen, cellulose, etc.) in the environment into fermentable components. Hence, the clostridia play an important role iature in biodegradation and the carbon cycle. In anaerobic clostridial infections, these enzymes play a role in invasion and pathology.
Most of the clostridia are saprophytes, but a few are pathogenic for humans, primarily Clostridium perfringens, C. difficile, C. tetani and C. tetani. Those that are pathogens have primarily a saprophytic existence iature and, in a sense, are opportunistic pathogens. Clostridium tetani and Clostridium botulinum produce the most potent biological toxins known to affect humans. As pathogens of tetanus and food-borne botulism, they owe their virulence almost entirely to their toxigenicity. Other clostridia, however, are highly invasive under certain circumstances.
Clostridium perfringens
Clostridium perfringens, which produces a huge array of invasins and exotoxins, causes wound and surgical infections that lead to gas gangrene, in addition to severe uterine infections. Clostridial hemolysins and extracellular enzymes such as proteases, lipases, collagenase and hyaluronidase, contribute to the invasive process. Clostridium perfringens also produces an enterotoxin and is an important cause of food poisoning. Usually the organism is encountered in improperly sterilized (canned) foods in which endospores have germinated.
Food poisoning
Clostridium perfringens is classified into 5 types (A�E) on the basis of its ability to produce one or more of the major lethal toxins, alpha, beta, epsilon and iota (α, β, ε, and ι). Enterotoxin (CPE)-producing (cpe+) C. perfringens type A is reported continuously as one of the most common food poisoning agents worldwide. An increasing number of reports also implicate the organism in 5%�15% of antibiotic�associated diarrhea (AAD) and sporadic diarrhea (SD) cases in humans, as well as diarrhea cases in animals.
Most food poisoning strains studied carry cpe in their chromosomes; isolates from AAD and SD cases bear cpe in a plasmid. Why C. perfringens strains with cpe located on chromosomes or plasmids cause different diseases has not been satisfactorily explained. However, the relatively greater heat resistance of the strains with chromosomally located cpe is a plausible explanation for these strains’ survival in cooked food, thus causing instances of food poisonings. The presence of C. perfringens strains with chromosomally located cpe in 1.4% of American retail food indicates that these strains have an access to the food chain, although sources and routes of contamination are unclear.
An explanation for the strong association between C. perfringens strains with plasmid-located cpe and cases of AAD and SD disease may be in vivo transfer of the cpe plasmid to C. perfringens strains of the normal intestinal microbiota. Thus, a small amount of ingested cpe+ C. perfringens would act as an infectious agent and transfer the cpe plasmid to cpe� C. perfringens strains of the normal microbiota. Conjugative transfer of the cpe plasmid has been demonstrated in vitro, but no data exist on horizontal gene transfer of cpe in vivo, and whether cpe+ strains that cause AAD and SD are resident in the gastrointestinal tract or acquired before onset of the disease is unknown.
Report of C. perfringens Food Poisoning
Clostridium perfringens is a common cause of outbreaks of foodborne illness in the United States, especially outbreaks in which cooked beef is the implicated source. This is a condensed version of an MMWR report that describes an outbreak of C. perfringens gastroenteritis following St. Patrick’s Day meals of corned beef. The report typifies outbreaks of C. perfringens food poisoning.
Report
On March 18, 1993, the Cleveland City Health Department received telephone calls from 15 persons who became ill after eating corned beef purchased from one delicatessen. After a local newspaper article publicized this problem, 156 persons contacted the health department to report onset of diarrheal illness within 48 hours of eating food from the delicatessen on March 16 or March 17. Symptoms included abdominal cramps (88%) and vomiting (13%); no persons were hospitalized. The median incubation period was 12 hours (range: 2-48 hours). Of the 156 persons reporting illness, 144 (92%) reported having eaten corned beef; 20 (13%), pickles; 12 (8%), potato salad; and 11 (7%), roast beef.
In anticipation of a large demand for corned beef on St. Patrick’s Day (March 17), the delicatessen had purchased 1400 pounds of raw, salt-cured product. Beginning March 12, portions of the corned beef were boiled for 3 hours at the delicatessen, allowed to cool at room temperature, and refrigerated. On March 16 and 17, the portions were removed from the refrigerator, held in a warmer at 120oF (48.8oC), and sliced and served. Corned beef sandwiches also were made for catering to several groups on March 17; these sandwiches were held at room temperature from 11 a.m. until they were eaten throughout the afternoon.
Cultures of two of three samples of leftover corned beef obtained from the delicatessen yielded greater than or equal to 105 colonies of C. perfringens per gram.
Following the outbreak, public health officials recommended to the delicatessen that meat not served immediately after cooking be divided into small pieces, placed in shallow pans and chilled rapidly on ice before refrigerating, and that cooked meat be reheated immediately before serving to an internal temperature of greater than or equal to 165oF (74 C).
Analysis
C. perfringens is a ubiquitous, anaerobic, Gram-positive, spore-forming bacillus and a frequent contaminant of meat and poultry. C. perfringens food poisoning is characterized by onset of abdominal cramps and diarrhea 8-16 hours after eating contaminated meat or poultry. By sporulating, this organism can survive high temperatures during initial cooking; the spores germinate during cooling of the food, and vegetative forms of the organism multiply if the food is subsequently held at temperatures of 60-125oF (16-52oC). If served without adequate reheating, live vegetative forms of C. perfringens may be ingested. The bacteria then elaborate the enterotoxin that causes the characteristic symptoms of diarrhea and abdominal cramping.
Laboratory confirmation of C. perfringens foodborne outbreaks requires quantitative cultures of implicated food or stool from ill persons. This outbreak was confirmed by the recovery of greater than or equal to 105 organisms per gram of epidemiologically implicated food. An alternate criterion is that cultures of stool samples from persons affected yield greater than or equal to 106 colonies per gram. Stool cultures were not done in this outbreak. Serotyping is not useful for confirming C. perfringens outbreaks and, in general, is not available.
Corned beef is a popular ethnic dish that is commonly served to celebrate St. Patrick’s Day. The errors in preparation of the corned beef in this outbreak were typical of those associated with previously reported foodborne outbreaks of C. perfringens. Improper holding temperatures are a contributing factor in most C. perfringens outbreaks reported to CDC. To avoid illness caused by this organism, food should be eaten while still hot or reheated to an internal temperature of greater than or equal to 165oF (74oC) before serving.
Gas gangrene
Gas gangrene generally occurs at the site of trauma or a recent surgical wound. The onset of gas gangrene is sudden and dramatic. About a third of cases occur on their own. Patients who develop this disease in this manner often have underlying blood vessel disease (atherosclerosis or hardening of the arteries), diabetes, or colon cancer.
Clostridium perfringens produces many different toxins, four of which (alpha, beta, epsilon, iota) can cause potentially deadly syndromes. The toxins cause damage to tissues, blood cells, and blood vessels.
Gas gangrene is marked by a high fever, brownish pus, gas bubbles under the skin, skin discoloration, and a foul odor. It is the rarest form of gangrene, and only 1,000 to 3,000 cases occur in the United States each year. It can be fatal if not treated immediately.
Clostridium difficile
Clostridium difficile causes antibiotic-associated diarrhea (AAD) and more serious intestinal conditions such as colitis and pseudomembranous colitis in humans. These conditions generally result from overgrowth of Clostridium difficile in the colon, usually after the normal intestinal microbiota flora has been disturbed by antimicrobial chemotherapy.
People in good health usually do not get C. difficile disease. Individuals who have other conditions that require prolonged use of antibiotics and the elderly are at greatest risk. Also, individuals who have recently undergone gastrointestinal surgery, or have a serious underlying illness, or who are immunocompromised, are at risk.
C. difficile produces two toxins: Toxin A is referred to as an enterotoxin because it causes fluid accumulation in the bowel. Toxin B is an extremely lethal (cytopathic) toxin.
Stool cultures for diagnosis of the bacterium may be complicated by the occurrence and finding of non toxigenic strains of the bacterium, so the most reliable tests involve testing for the presence of the Toxin A and/or Toxin B in the stool. The toxins are very unstable. They degrade at room temperature and may be undetectable within two hours after collection of a stool specimen leading to false negative results of the diagnosis.
In the hospital and nursing home setting, C. difficile infections can be minimized by judicious use of antibiotics, use of contact precautions with patients with known or suspected cases of disease, and by implementation of an effective environmental and disinfection strategy.
Clostridium difficile infections can usually be treated successfully with a 10-day course of antibiotics including metronidiazole or vancomycin (administered orally).
Clostridium tetani
Clostridium tetani is the causative agent of tetanus. The organism is found in soil, especially heavily-manured soils, and in the intestinal tracts and feces of various animals. Carrier rates in humans vary from 0 to 25%, and the organism is thought to be a transient member of the flora whose presence depends upon ingestion. The organism produces terminal spores within a swollen sporangium giving it a distinctive drumstick appearance. Although the bacterium has a typical Gram-positive cell wall, it may stain Gram-negative or Gram-variable, especially in older cells.
Tetanus is a highly fatal disease of humans. Mortality rates reported vary from 40% to 78%. The disease stems not from invasive infection but from a potent neurotoxin (tetanus toxin or tetanospasmin) produced when spores germinate and vegetative cells grow after gaining access to wounds. The organism multiplies locally and symptoms appear remote from the infection site.
Because of the widespread use of the tetanus toxoid for prophylactic immunization, fewer than 150 cases occur annually in the U.S., but the disease is a significant problem world-wide where there are >300,000 cases annually. Most cases in the U.S occur in individuals over age 60, which is taken to mean that waning immunity is a significant risk factor.
Pathogenesis of tetanus
Most cases of tetanus result from small puncture wounds or lacerations which become contaminated with C. tetani spores that germinate and produce toxin. The infection remains localized, often with only minimal inflammatory damage. The toxin is produced during cell growth, sporulation and lysis. It migrates along neural paths from a local wound to sites of action in the central nervous system. The clinical pattern of generalized tetanus consists of severe painful spasms and rigidity of the voluntary muscles. The characteristic symptom of “lockjaw” involves spasms of the masseter muscle. It is an early symptom which is followed by progressive rigidity and violent spasms of the trunk and limb muscles. Spasms of the pharyngeal muscles cause difficulty in swallowing. Death usually results from interference with the mechanics of respiration.
Sir to as opisthotonos and risus sardonicus. Original in the Royal College of Surgeons of Edinburgh, Scotland.
Neonatal tetanus accounts for about half of the tetanus deaths in developing countries. In a study of neonatal mortality in Bangladesh, 112 of 330 infant deaths were due to tetanus. Neonatal tetanus follows infection of the umbilical stump in infants born to nonimmune mothers (therefore, the infant has not acquired passive immunity). It usually results from a failure of aseptic technique during birthing procedures, but certain cultural practices may contribute to infection.
Tetanus Toxin
There have been 11 strains of C. tetani distinguished primarily on the basis of flagellar antigens. They differ in their ability to produce tetanus toxin (tetanospasmin), but all strains produce a toxin which is identical in its immunological and pharmacological properties. Tetanospasmin is encoded on a plasmid which is present in all toxigenic strains.
Tetanus toxin is one of the three most poisonous substances known to humans, the other two being the toxins of botulism and diphtheria. The toxin is produced by growing cells and released only on cell lysis. Cells lyse naturally during germination the outgrowth of spores, as well as during vegetative growth. After inoculation of a wound with C. tetani spores, only a minimal amount of spore germination and vegetative cell growth are required until the toxin is produced.
The bacterium synthesizes the tetanus toxin as a single 150kDa polypeptide chain (called the progenitor toxin), that is cleaved extracellularly by a bacterial protease into a 100 kDa heavy chain (fragment B) and a 50kDa light chain (fragment A), which remain connected by a disulfide bridge. The specific protease that cleaves the progenitor toxin can be found in culture filtrates of C. tetani. Cleavage of the progenitor toxin into A and B fragments can also be induced artificially with trypsin.
Tetanus toxin is produced in vitro in amounts up to 5 to 10% of the bacterial weight. Because the toxin has a specific affinity for nervous tissue, it is referred to as a neurotoxin. The toxin has no known useful function to C. tetani. Why the toxin has a specific action oervous tissue, to which the organism naturally has no access, may be an anomaly of nature. The toxin is heat labile, being destroyed at 56oC in 5 minutes, and is O2 labile. The purified toxin rapidly converts to toxoid at 0oC in the presence of formalin.
Toxin Action
Tetanospasmin initially binds to peripheral nerve terminals. It is transported within the axon and across synaptic junctions until it reaches the central nervous system. There it becomes rapidly fixed to gangliosides at the presynaptic inhibitory motor nerve endings, and is taken up into the axon by endocytosis. The effect of the toxin is to block the release of inhibitory neurotransmitters (glycine and gamma-amino butyric acid) across the synaptic cleft, which is required to inhibit nervous impulse. If nervous impulses cannot be checked by normal inhibitory mechanisms, it produces the generalized muscular spasms characteristic of tetanus. Tetanospasmin appears to act by selective cleavage of a protein component of synaptic vesicles, synaptobrevin II, and this prevents the release of neurotransmitters by the cells.
The receptor to which tetanospasmin binds has been reported as ganglioside GT and/or GD1b, but its exact identity is still in question. Binding appears to depend on the number and position of sialic acid residues on the ganglioside. Isolated B fragments, but not A fragments, will bind to the ganglioside. The A fragment has toxic (enzymatic) activity after the B fragment secures its entry. Binding appears to be an irreversible event so that recovery depends on sprouting a new axon terminal.
Immunity
Unlike other toxigenic diseases, such as diphtheria, recovery from the natural disease usually does not confer immunity, since even a lethal dose of tetanospasmin is insufficient to provoke an immune response.
Prophylactic immunization is accomplished with tetanus toxoid, as part of the DPT (DTaP) vaccine or the DT (TD) vaccine. Three injections are given in the first year of life, and a booster is given about a year later, and again on the entrance into elementary school.
Whenever a previously-immunized individual sustains a potentially dangerous wound, a booster of toxoid should be injected. Wherever employed, intensive programs of immunization with toxoid have led to a striking reduction in the incidence of the disease.
Clostridium botulinum
C. botulinum
C. botulinum is a large anaerobic bacillus that forms subterminal endospores. It is widely distributed in soil, sediments of lakes and ponds, and decaying vegetation. Hence, the intestinal tracts of birds, mammals and fish may occasionally contain the organism as a transient. Seven toxigenic types of the organism exist, each producing an immunologically distinct form of botulinum toxin. The toxins are designated A, B, C1, D, E, F, and G). In the U.S., type A is the most significant cause of botulism, involved in 62% of the cases. Not all strains of C. botulinum produce the botulinum toxin. Lysogenic phages encode toxin serotypes C and D, and non lysogenized bacteria (which exist iature) do not produce the toxin. Type G toxin is thought to be plasmid encoded.
Pathogenesis of Botulism
Food-borne Botulism
In food-borne botulism, the botulinum toxin is ingested with food in which spores have germinated and the organism has grown. The toxin is absorbed by the upper part of the GI tract in the duodenum and jejunum and passes into the blood stream by which it reaches the peripheral neuromuscular synapses. The toxin binds to the presynaptic stimulatory terminals and blocks the release of the neurotransmitter acetylcholine which is required for a nerve to simulate the muscle.
Food-borne botulism is not an infection but an intoxication since it results from the ingestion of foods that contain the preformed clostridial toxin. In this respect, it resembles staphylococcal or Bacillus cereus food poisoning. Botulism results from eating uncooked foods in which contaminating spores have germinated and produced the toxin. C. botulinum spores are relatively heat resistant and may survive the sterilizing process of improper canning procedures. The anaerobic environment produced by the canning process may further encourage the outgrowth of spores. The organisms grow best ieutral or “low acid” vegetables (>pH4.5).
Clinical symptoms of botulism begin 18-36 hours after toxin ingestion with weakness, dizziness and dryness of the mouth. Nausea and vomiting may occur. Neurologic features soon develop, including blurred vision, inability to swallow, difficulty in speech, descending weakness of skeletal muscles and respiratory paralysis.
Botulinum toxin may be transported withierves in a manner analogous to tetanospasmin, and can thereby gain access to the CNS. However, symptomatic CNS involvement is rare.
Infant Botulism
Infant botulism is due to infection caused by C. botulinum. The disease occurs in infants 5 – 20 weeks of age that have been exposed to solid foods, presumably the source of infection (spores). It is characterized by constipation and weak sucking ability and generalized weakness. C. botulinum can apparently establish itself in the bowel of infants at a critical age before the establishment of competing intestinal microbiota. Production of toxin by bacteria in the GI tract induces symptoms. This “infection-intoxication” is restricted to infants. C. botulinum organisms, as well as toxin, can be found in the feces of infected infants. Almost all known cases of the disease have recovered. The possible role of infant botulism in “sudden infant death syndrome-SIDS” has been suggested but remains unproven. C. botulinum, its toxin, or both have been found in the bowel contents of several infants who have died suddenly and unexpectedly.
The Botulinum Toxins
The botulinum toxins are very similar in structure and function to the tetanus toxin, but differ dramatically in their clinical effects because they target different cells in the nervous system. Botulinum neurotoxins predominantly affect the peripheral nervous system reflecting a preference of the toxin for stimulatory motor neurons at a neuromuscular junction. The primary symptom is weakness or flaccid paralysis. Tetanus toxin can affect the same system, but the tetanospasmin shows a tropism for inhibitory motor neurons of the central nervous system, and its effects are primarily rigidity and spastic paralysis.
Botulinum toxin is synthesized as a single polypeptide chain with a molecular weight around 150 kDa. In this form, the toxin has a relatively low potency. The toxin is nicked by a bacterial protease (or possibly by gastric proteases) to produce two chains: a light chain (the A fragment) with a molecular weight of 50 kDa; and a heavy chain (the B fragment), with a mw of 100kDa. As with tetanospasmin, the chains remain connected by a disulfide bond. The A fragment of the nicked toxin, on a molecular weight basis, becomes the most potent toxin found iature.
Toxin Action
The botulinum toxin is specific for peripheral nerve endings at the point where a motor neuron stimulates a muscle. The toxin binds to the neuron and prevents the release of acetylcholine across the synaptic cleft.
The heavy chain of the toxin mediates binding to presynaptic receptors. The nature of these receptors is uncertain; different toxin types seem to utilize slightly different receptors. The binding region of the toxin molecule is located near the carboxy terminus of the heavy chain. The amino terminus of the heavy chain is thought to form a channel through the membrane of the neuron allowing the light chain to enter. The toxin (A fragment) enters the cell by receptor mediated endocytosis. Once inside a neuron, different toxin types probably differ in mechanisms by which they inhibit acetylcholine release, but a mechanism similar to or identical to tetanospasmin has been reported (i.e., proteolytic cleavage of synaptobrevin II). The affected cells fail to release a neurotransmitter, thus producing paralysis of the motor system. Once damaged, the synapse is rendered permanently useless. The recovery of function requires sprouting of a new presynaptic axon and the subsequent formation of a new synapse.
As stated above, the mechanism by which acetylcholine release is prevented is not known. However, recent evidence suggests that both botulinum toxin as well as tetanus toxin are zinc-dependent endopeptidases that cleave specific proteins that are involved in excretion of neurotransmitters. Both toxins cleave a set of proteins called synaptobrevins. Synaptobrevins are found in synaptic vesicle of neurons, the vesicles responsible for release of neurotransmitters. Presumably, proteolytic cleavage of synaptobrevin II would interfere with vesicle function and release of neurotransmitters.
Immunity
On the average there are about 25 cases of botulism annually in the United States. Prior to the advent of critical care, the case fatality rate exceeded 60%, but currently it is about 20%. The first (or only) patient in an outbreak has a 25% chance of death, whereas subsequent cases which are diagnosed and treated more quickly, carry only a 4% risk.
Each od the toxins that cause botulism is specifically neutralized by its antitoxin. Botulinum toxins can be toxoided and make good antigens for inducing protective antibody. As with tetanus, immunity to botulism does not develop, even with severe disease, because the amount of toxiecessary to induce an immune response is lethal. Repeated occurrences of botulism has been reported.
Once the botulinum toxin has bound to nerve endings, its activity is unaffected by antitoxin. Any circulating (“unfixed”) toxin can be neutralized by intravenous injection of antitoxin. Therefore, individuals known to have ingested food with botulism should be treated immediately with antiserum.
A multivalent toxoid evokes good protective antibody response but its use is unjustified due to the infrequency of the disease. An experimental vaccine exists for laboratory workers.
Prevention
The most important aspect of botulism prevention is proper food handling and preparation. The spores of C. botulinum can survive boiling (100oC at 1 atm) for more than one hour, although they are killed by autoclaving. Because the toxin is heat-labile, boiling or intense heating (cooking) of contaminated food will inactivate the toxin. Food containers that bulge may contain gas produced by C. botulinum and should not be opened or tasted. Other foods that appear to be spoiled should not be tasted.
Botulism and Bioterrorism
Botulinum Toxin in Biowarfare……of course, it has been thought of …….botulinum toxin is the most potent poison known for humans; 10 grams is a lethal dose for the human population of Los Angeles. Below is an interesting anecdote that appeared in JAMA Vol. 285, No. 21, June 6, 2001
To the Editor:
A historical incident illustrates a number of features of botulinum toxiot discussed in the review of bioweaponry by Dr. Arnon and colleagues.
During World War II, the US Office of Strategic Services (OSS) developed a plan for Chinese prostitutes to assassinate high-ranking Japanese officers with whom they sometimes consorted in occupied Chinese cities. Concealing traditional weapons on the women at the appropriate time would obviously be difficult. Therefore, under the direction of Stanley Lovell, the OSS prepared gelatin capsules “less than the size of the head of a common pin” containing a lethal dose of botulinum toxin. Wetted, a capsule could be stuck behind the ear or in scalp hair, later to be detached and slipped into the officer’s food or drink. The OSS recognized that the normal background of botulism cases would deflect suspicion from the women.
The capsules were shipped to Chunking, China. The Navy detachment there, taking nothing for granted, tested the capsules on stray donkeys. The donkeys lived. Lovell was informed that the capsules were faulty, and the project was abandoned. Much later, Lovell learned of the donkey test with, one imagines, some consternation, since “donkeys are one of the few living creatures immune to botulism.”
This incident has been retold in other publications. No source for the donkey-resistance information is ever given. More recent experience shows that botulism can occur in mules and donkeys (R. H. Whitlock, DVM, PhD, written communication, April 27, 2001).
