Spore forming gram positive Bacilli: Bacillus and Clostridia Species
Dr. Y. J. Peter (MB BCh, MSc, FMCPath)
drjonahp@yahoo.com
Lecture Outline
Introduction
Bacillus species
Pathogenesis
Laboratory diagnosis
Clostridium species
Pathogenesis
Laboratory diagnosis
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Introduction
The gram-positive spore-forming bacilli are the Bacillus and Clostridium species.
These bacilli (rod-like) are ubiquitous, and because they form spores, they can survive in the environment for many years.
They are divided into two groups according to their oxygen requirement.
Bacillus species are aerobes and the Clostridium species are anaerobes.
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Introduction 2
Most species of Bacillus and related genera, do not cause disease. There are a few species, however, that cause important diseases in humans.
Anthrax, a classical disease in the history of microbiology, is caused by Bacillus anthracis. Anthrax remains an important disease of animals and occasionally of humans. Because of its potent toxins, B anthracis is a major potential agent of bioterrorism and biologic warfare. Bacillus cereus and Bacillus thuringiensis cause food poisoning and occasionally eye or other localized infections.
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Introduction 3
The genus Clostridium is extremely heterogeneous, and more than 200 species have been described.
Clostridia cause several important toxin-mediated diseases, including tetanus (Clostridium tetani), botulism (Clostridium botulinum), gas gangrene (Clostridium perfringens), and antibiotic-associated diarrhea and pseudomembranous colitis (Clostridium difficile).
Other clostridia are also found in mixed anaerobic infections in humans.
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Bacillus species
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Bacillus species
Ubiquitous and found worldwide, its normal habitat is the soil or contaminated vegetative material.
It is a gram-positive bacilli, but can be gram-variable, often hemolytic and motile (except B anthracis).
It is endospore-forming, aerobic or facultative anaerobic.
Resistant to radiation, chemicals, heat, desiccation (dipicolinic acid). Steam autoclaving is necessary for its destruction.
Koch’s postulates was first applied to B anthracis.
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Bacillus species
It is a large rectangular bacilli (that occurs in chains) with central, oval spores. It is non-motile forming large, waxy (dry) colonies, irregular shape, not haemolytic.
It is aerobic and grows well at 37°C. On nutrient agar, it produces filamentous structures called “Medusa” head colonies.
Requires thymine and other amino acids for growth. Ferments Glucose, Sucrose, Maltose and Trehalose.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Medusa head appearance
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Antigens and Virulence Factors
The capsule is an important virulence factor, it inhibits phagocytosis. It is found in clinical specimens but not always on laboratory media.
The exotoxin is a protein complex that increases vascular permeability leading to shock.
Virulence is dependent on the presence of two Plasmids. One codes for the capsule, while the other codes for the toxin. This plasmid must be present in a Bacillus strain for it to be virulent.
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Epidemiology
Anthrax is primarily a disease of herbivores.
Human infection is by contact with infected animals or contaminated animal products. This is rampant in countries where animals are not vaccinated. The disease is rare where animals are vaccinated.
Recent interest in this bacteria arose because of its use as a terrorism agent in the USA.
Human anthrax maybe acquired by
1) Inoculation, 2) Ingestion or 3) Inhalation.
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Clinical Infections
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Bacillus anthracis (Anthrax)
This condition is enzootic in the middle east. It is transmitted to humans by contact with animals or animal products. The spores remain infectious in soil for many years.
Usually causes cutaneous inoculation which results in a slow healing ulcer, the bacilli may spread to the lymphatics and bloodstream. It is associated with 20% mortality if untreated.
Respiratory anthrax infection is usually fatal even if treated.
Gastrointestinal anthrax infection occurs in some Asian countries
It may be used as a biological warfare agent.
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Pathogenesis of Anthrax infection
Poly-D-glutamic acid (protein) capsule which is antiphagocytic, has a 3-component exotoxin
Protective antigen (PA) which binds to cells, forming channels that permits EF and LF entry.
Edema factor (EF), adenyl cyclase causes fluid to accumulate at the site of infection and inhibit immune function.
Lethal factor (LF), disrupts cell's functions, stimulates TNF-alpha and IL-1-beta, leading to death of the infected cells.
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Cutaneous anthrax-Malignant pustule
Inoculation is the most common mode of infection. Spores enter through the skin from contaminated soil or infected animal products (hides, goat hair and wool). Infections occur mainly on exposed areas of the skin. Initially as a painless papule at the site of scratch. The papule progresses to an ulcer surrounded by vesicles and then to a necrosis due to effect of the toxin described as black ‘Eschar’. There is lymphadenopathy and massive edema. If untreated the bacteria invades regional lymph nodes and the blood stream, this is usually lethal. Mortality rate without treatment is about 20%.
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Pulmonary anthrax-Wool sorter’s disease
Transmission is by inhalation. This is also the most likely route for biological weapons. Patients may remain asymptomatic for a prolonged period (2 months) different from other forms of disease. Spores may be transmitted from dust, wool fibers or brushes made from animal hair.
Symptoms include fever, dyspnea, cough, headache, vomiting, chills, chest and abdominal pain. Bacteria multiply in the bronchi then spreads to lungs, lymphatics and blood stream. This produces intense inflammation, hemorrhage and septicemia as bacteria multiply. It has a high mortality rate (requires very rapid treatment).
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Gastrointestinal anthrax
This is a rare mode of transmission. However outbreaks follow ingestion of spores from contaminated meat.
Clinical symptoms depend on the site of infection. Invasion of upper intestinal tract results in formation of ulcers in mouth or esophagus and regional lymphadenopathy, edema and sepsis.
Bacteria in the caecum or terminal ileum results in symptoms of nausea, vomiting and malaise which progress to systemic disease. It results in a hemorrhagic diarrhea and death.
Mortality rate is as high as 100%.
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Laboratory diagnosis
Specimen: Skin or fluid from vesicles, sputum, feces or blood.
Microscopy: Gram-positive, large rectangular bacilli. Stains from cultures show long chains with spores.
Fluorescent Staining: for rapid detection.
Culture: Blood agar shows large, waxy, irregular, grey colonies. Nutrient agar show “Medusa” head colonies.
Confirmatory Tests: Non-motile, Gelatin liquification.
PCR for rapid detection.
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Bacillus anthracis
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Treatment, Control and Prevention
Drug of first choice is Ciprofloxacin. Alternative drugs include Penicillin, Erythromycin, Tetracyclines or Chloramphenicol after suitable anti-microbial testing.
These same drugs are used for the control of the infection.
Prevent transmission from infected animals.
Prevent transmission from infected cases by isolation of patients and use of protective clothing. Chemoprophylaxis using ciprofloxacin for those at risk. Vaccines are available.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Bacillus cereus
This is a cause of food poisoning.
Morphology and characteristics of the organism is similar to Bacillus anthracis.
It differs from it in that it is motile and lacks a glutamic acid capsule.
It is a saprophyte that resides on vegetation, soil and water.
The spores are heat resistant and most strains produce toxins.
It is associated with foods such as cereals and especially rice. Produces food-borne intoxication. Frequently associated with “Chinese foods”.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Bacillus cereus
The organism produces two distinct diseases. Clinical symptoms include:
Heat-stable enterotoxin (emetic form) or
Heat-labile enterotoxin (diarrheal form). This is similar to V cholera – stimulates cAMP watery diarrhea from contaminated rice, meat or vegetables.
Other Clinical conditions includes ocular infections after trauma > necrotic toxin> hemolysin> phospholipase C.
Intravenous catheter related sepsis, endocarditis or meningitis.
Immunocompromised persons and drug abusers are most at risk.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Bacillus cereus
Emetic Disease:
Vomiting 1-6 hours after ingestion, due to the action of preformed toxin (intoxication) in the meal.
It has a low molecular weight, stable to heat, acid and with enzymatic activity.
It is associated with cereals and rice.
Symptoms: Nausea, vomiting and abdominal cramps.
Fever and diarrhea are usually absent.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Bacillus cereus
Diarrhoeal Disease:
Diarrhea about 8-24 hours after ingestion of contaminated meat, vegetables and sauces as a result of the action of enterotoxins which is heat labile.
Formed in the intestine following ingestion of a meal. These toxins are similar to toxins produced by S typhi and E coli.
Symptoms include diarrhea, nausea and abdominal cramps.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Laboratory Diagnosis
Specimen: Suspected food if available.
Feces is not useful because colonisation is common. Fecal isolation in a cluster of cases may be useful.
Microscopy: Gram-positive bacilli with spores.
Culture: Blood agar.
Shows a large number (>108) of organisms present per gram of food.
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Treatment and control
This is a self-limiting disease.
Treatment is fluid and electrolyte replacement in serious cases. Antibiotics is not required.
Control: Only consume freshly prepared meals. Ideally eat only freshly prepared foods.
Disease is prevented by proper cooling and storage of prepared foods.
Adequate heating when using leftover foods.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clostridium species
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Clostridium species
These are large, gram-positive, straight or slightly curved organisms with rounded ends which are pleomorphic. They produce spores and are anaerobic. They inhabit the soil and intestinal tract of humans and animals. There are two types:
1. Saccharolytic species which ferment carbohydrates to produce acid and
2. Proteolytic species which metabolise proteins yielding foul smelling amines.
Most species produce disease by the action of enzymes and toxins (they are not invasive) but gas gangrene is invasive.
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Clostridium species
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Clostridium species
The clostridia are an opportunistic pathogen. Nonetheless, they are responsible for some of the deadliest diseases including gas gangrene, tetanus and botulism.
Less life-threatening diseases include pseudomembranous colitis (PC) and food poisoning.
They cause disease primarily through the production of numerous exotoxins.
There are four important members to this group.
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Clostridium species
• Clostridium tetani
• Clostridium botulinum
• Clostridium perfringens
• Clostridium difficile
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Clostridium tetani
Causes tetanus or “lock jaw” syndrome.
Has a “drumstick” appearance (terminal spores). With peritrichious flagella, it is highly motile. Spores enter the patient through wounds, superficial abrasions, body puncture, gunshots, burns or animal bite. Post-operative wounds and into baby's umbilical stump (“Tetanus neonatarum”).
It is not an invasive disease, remains in localised areas of tissue. It requires anaerobic environment to germinate and grow. Many serotypes produce same exotoxin. Immunity directed against exotoxin. Toxin producing strains have large plasmids. Loss of plasmid converts to a non-virulent strain.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clostridium tetani
Pathogenesis:
Two toxins act in concert: Tetanospasmin and Tetanolysin
1. Binds to cells at the neuromuscular junctions.
2. Crosses nerve cell membrane and is transported to inhibitory interneurons.
3. Blocks release of inhibitory transmitters, γ-amino-butyric acid (GABA) and glycine.
4. Toxin is transmitted by motor nerves and nerve fibres. Also through blood flow to the CNS.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clostridium tetani infection
1. Generalised tetanus is the most common presentation.
Incubation period is variable with distance of wound from the brain.
Involves masseter muscles with typical sardonic smile resulting from sustained muscle contraction. Other signs include drooling, sweating, irritability and muscle spasms, involving neck and jaws mucles. This disease is called “lockjaw” syndrome.
Death occurs by muscular spasms preventing the respiratory muscle action.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clostridium tetani infection
2. Localised tetanus: Confined to the musculature of the limb at primary site of infection.
3. Cephalic tetanus: In which the primary site of infection is the head. The prognosis is very poor.
4. Neonatal tetanus: “Tetanus neonatarum” –
Results from umbilical stump infection can lead to generalised tetanus. Mortality rate is more than 90%.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Laboratory diagnosis
The nature of the clinical presentation is important.
Specimen: From primary site of infection.
Microscopy: Gram-positive, “drumstick” like appearance.
Culture: Blood agar anaerobic incubation. Difficult if only few bacteria are in lesions, it is easily killed by exposure to air.
Inoculate in Cooked Meat Broth.
Confirmatory test: After isolation, confirm by detection of toxin using anti-toxin neutralisation test.
Virulence in animals: Used to test the toxin.
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Treatment and Prevention
1. Surgical wound debridement to aerate the site.
2. Metronidazole eliminates vegetative bacteria.
3. Passive immunisation with tetanus immunoglobulin (anti-toxic serum - ATS), specially in un-vaccinated individuals or those without boosters.
4. Active immunisation: Vaccination with tetanus toxoid (TT -DPT). This is made up of 3 doses of formalin inactivated tetanus toxoid at 2, 4 and 6 months of age. Boosters are given at 18 months and then followed by boosters every 5-10 years.
Giving this is important for military personnel, females prior to pregnancy and children at school age.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clostridium perfringens
The causative agent of gas gangrene. Other species sometimes involved are C novyi, C histolyticum and C septicum. These are large, rectangular, gram-positive bacilli. It is non-motile.
The spores are subterminal, not bulging, and are rarely found in-vivo or in-vitro isolation.
Culture: Form hemolytic colonies on blood agar. Ferments carbohydrates and produces gas. Produces at least 12 different toxins and numerous enzymes as virulence factors.
Diseases is caused by:
1) Severe necrotising wound infection, 2) Self-limiting gastroenteritis and 3) Severe haemorrhagic diarrhea.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clostridium perfringens
There are 12 different toxins and enzymes named using Greek letters; α, β …., α-toxin or lecithinase is the most important. Causes cell membrane hydrolysis and cell destruction. Other enzymes include; collagenase, hyaluronidase, fibrinolysin, DNase and neuraminidase.
C perfringens is sub-divided into five serotypes. A, B, C, D and E. Type A is most common, (also found in gas gangrene). Type A produces a heat labile “enterotoxin”.
Type C produces severe life-threatening diarrhea. Affects ion transport in the intestine and membrane permeability.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clostridium perfringens
Infection follows contamination of wounds with spores (invasive infection) in damaged tissue. Wounds could be result of military casualties, automobile or farm equipment accidents. Bacteria and spores are found in human and animal excreta.
Spores indirectly derived from dirty clothing, street dust or air in hospital operating theatres from poorly designed ventilation system. Bacteria germinate then secrete exotoxins and enzymes cause tissue destruction. Disease spreads rapidly in a necrotic environment. Bacteria ferment carbohydrates and produce gas in tissues.
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Pathogenesis
Pressure from gas causes restriction of blood supply and necrosis. In the absence of surgical and anti-toxic treatment, severe toxaemia and death frequently occur. Clostridium perfringens can also cause diffuse spreading cellulitis accompanied by toxemia.
Abdominal infections may originate from the large intestine either from a bowel perforation or contaminated injection site. Perineal infections from intestinal organisms more common in hospitalised patients.
Clostridium perfringens in female genital tract are a normal flora. But this can induce abortions and uterine gas gangrene.
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Clinical presentation
Myonecrosis or gas gangrene is life threatening. Symptoms include intense pain, about one week after infection. Followed by extensive muscle necrosis, shock, renal failure and death often within 48 hours of onset.
Examination reveals necrotic tissue and presence of gas due to metabolic activity of rapidly dividing bacteria. Toxins cause extensive haemolysis and bleeding.
Other Soft tissue infections include: Cellulitis, Fasciitis or suppurative myositis.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clinical presentation
Food Poisoning (this is a common disease). Produced by C perfringens type A, incubation period is 8-24 hours.
Symptoms: Abdominal cramps and watery diarrhea, but no fever, nausea or vomiting. Lasts about 24 hours. Associated with meat and meat products containing spores of C perfringens type A.
Prevention: Refrigerate prepared food to prevent production of enterotoxin. Adequate heating of food can destroy the toxin.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clinical presentation
Necrotising Enteritis (Rare, acute necrotising process in intestine). Produced by biovar type C
Symptoms: Abdominal pain, bloody diarrhea, shock and peritonitis. Mortality rate is 50%.
Risk factors include exposure to large numbers of bacteria and malnutrition.
Sepsis: This is a systemic infection involving the whole body. You must consider the significance of isolate (it may be a contaminant from the skin).
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Laboratory Diagnosis
Specimens : Exudate or biopsy material.
Microscopy : Gram-positive bacilli in specimens.
Culture : Anaerobic culture on blood agar. Inoculate cooked meat broth and sub-culture. Confirmatory tests:
1. Nagler reaction: α-toxin “lecithinase” breaks down lecithin in egg yolk media shown by an “opaque” area around the colony. Presence of anti-toxin on the media stops the action of the toxin.
2. Stormy fermentation in milk. Coagulated milk “blown apart” by gas produced due to lactose fermentation.
3.Biochemical tests can be used to identify individual species.
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Stormy fermentation in Litmus milk
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Treatment and Control
Surgical cleansing of wounds is most important. Anti-toxin may be used when complete surgical debridement is not possible. Penicillin is only useful in tissue which has blood supply.
Hyperbaric oxygen chambers: Infected tissue placed in chamber containing oxygen under pressure have some success in stopping growth of these organisms. Prognosis is very poor.
Mortality rate is betwen 40-100% in severe infections.
Control is difficult because of the ubiquitous distribution of this organism.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clostridium botulinum
Clostridium botulinum found in soil and water globally is a causative agent of “botulinum” food poisoning in canned or preserved food. It is a heterogenous group, fastidious, spore forming and an anaerobic bacilli. Spores are oval, bulging and sub-terminal, are motile with peritrichious flagella. Grows best at between 35-37°C but Some strains may grow and produce toxin at low temperatures. Spores survive boiling for hours and are only Killed by autoclaving.
Seven antigenically distinct toxins A-G exist. Human disease is caused by A, B, E and F.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Pathogenesis
Botulism neurotoxin is the most poisonous natural occurring toxin. One mililitre of culture fluid is lethal for 2 million mice and 1µg lethal for humans. Preformed neurotoxin is absorbed from GIT.
After absorption into blood stream it binds irreversibly to presynaptic nerve endings of peripheral nervous system and cranial nerves where it inhibits (neurotransmitter) acetylcholine release.
Also produces a binary toxin with two components that affect vascular permeability.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clinical presentation
1. Foodborne botulism.
Neurotoxin is present in preserved foods (often canned). Insufficient heating is an important cause in home canning. Types A and B in home-canned foods and type E in preserved fish.
Food does not look spoiled but a small taste causes symptoms. Incubation period is 1-2 days (or longer).
Smoked, salted or spiced meat also involved.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clinical presentation
Usually it starts with oculomotor muscles affected. Initial symptoms; blurred vision, diplopia, drooping eye lids, dilated pupils, dry mouth, nausea, vomiting, constipation and abdominal pain. Problems in speech and swallowing.
Signs of “bulbar” paralysis are progressive with weakness and sleepiness.
Death is due to respiratory or cardiac failure.
Mortality rate was as high as 70% now reduced to 10%.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clinical presentation
2. Infant botulism:
Bacteria are from the environment or food. Honey is sometimes associated with this disease. The absence of normal flora in the GIT of babies helps colonisation. Neurotoxin is produced in GIT of infants (different from foodborne disease). Disease affects children less than 1 yr (usually 1-6 months). Symptoms are non-specific include constipation, weak cry and “failure to thrive”. Disease progress to flaccid paralysis and respiratory arrest. “Floppy child syndrome”. Some infant deaths, may be a result of botulism.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clinical presentation
3. Wound Botulism:
Wound botulism may occur in a not always serious wounds. Even minor wounds needs to be considered in patients with typical symptoms.
Symptoms are blurred vision, weakness and difficulty in swallowing. Abdominal symptoms are less pronounced than in food borne infections.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Laboratory Diagnosis
Specimen: Food sample, faeces or vomit. Wound exudate or serum. Detect bacteria or toxin.
Microscopy: Typical appearance.
Culture: Anaerobic culture on blood agar.
Confirmatory tests: Lipase production, Hydrolyse Gelatin, Ferment Glucose and digest milk products.
Toxin detection: Toxin antitoxin neutralisation test. Detect toxin in food or in patient’s blood. Administered to mice with and without anti-toxin. Small amounts of toxin are lethal for mice. Care should be taken in carrying out tests because of virulence of toxin.
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Treatment
1. Remove unabsorbed toxin from stomach and intestines.
2. Neutralise unfixed toxin. In suspected cases administered anti-toxin ( polyvalent / types A, B and E) it does not neutralise fixed toxin but free residual toxin.
Infants are not administered anti-toxin because horse serum derived vaccine may produce hypersensitivity.
3. Administer metronidazole and penicillin.
4.Administer relevant intensive care (ventilation) and support.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Control
Avoid home canning. Home canned vegetables should be boiled for 10 min.
Sterilised home canned (pressure cook) vegetables should be maintained at the high temperature for 15 min.
Outbreaks from variety of foods reported specially when food is pre-cooked and stored at ambient temperature for 1-2 days.
Prophylactic dose of anti-toxin to be given to suspected cases.
Active immunization: Administer toxoid preparation in 3 doses, over 2 months. Produces solid immunity but routine usage not warranted. Recommended for laboratory workers.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Clostridium difficile
It is motile, gram-positive, oval, sub-terminal spores found in faeces of neonates and babies until weaning. Not a usual commensal in adults.
Evidence suggests organisms from exogenous source may enter patients who’s intestinal colonisation resistance has been affected by antibiotic usage.
Clostridium difficile is a normal flora (endogenous) in some adults (10%) and when other antibiotic sensitive organisms are eliminated from intestine these grow in large numbers to produce disease.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Pathogenesis�
Clostridium difficile produces antibiotic associated gastrointestinal diseases ranging from mild self-limiting diarrhea to severe life threatening ‘pseudomembranous colitis’. Diarrhea may be watery or bloody with abdominal cramps, leukocytosis and fever.
Antibiotics associated are mainly Clindamycin and Ampicillin. Some Cephalosporins are also implicated and, but there is virtually no antibiotic not implicated. Two toxins are produced;
1. Toxin A: Potent Enterotoxin produces diarrhea and has cytotoxic activity binds to brush border membranes of the gut at receptor sites.
2. Toxin B: Potent Cytotoxin. Has lethal effects.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Laboratory diagnosis
Specimen: feaces collected for microscopy
Culture: on selective media – Blood agar.
Confirmatory test: aims to demonstrate the presence of toxin.
Both toxins can be detected in feacal samples.
Cytotoxin (toxin B) shows in tissue culture cells.
Enterotoxin (toxin A) detected by immuno-assays.
Commercial kits using latex agglutination or ELISA tests are available for detecting both toxins.
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Treatment
1. Discontinue use of implicated antibiotic. Most patients recover spontaneously.
2. Suppress growth and toxin production by oral vancomycin and metronidazole.
3. Relapses are frequent in 20-30% cases. Only vegetative forms of bacteria killed by antibiotic. Spores are resistant. Second course of treatment with same antibiotic often successful.
Control is difficult because bacteria commonly exists in hospitals especially in areas near infected patients. Spores are difficult to destroy hence bacteria may survive in environment for months and is a major cause of nosocomial outbreaks.
Dr. J. Y. Peter (BM BCh, MSc, FMCPath)
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Thank you
Thank you for your attention
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