Clostridium botulinum: Biology and Virulence

Clostridium

• Gram-positive, spore-forming anaerobic bacilli.
• Usually found in soil, an exogenous source.
• Present as normal flora with lower oxygen concentration in the lower intestinal tract.
• Infections include: tetanus, gas gangrene , botulism, food poisoning, and pseudomembranous colitis.

Clostridium botulinum

• Etiologic agent of botulism.
• Collection of fastidious, spore-forming, anaerobic microorganisms that are large.
• There are different complex nutritional and environmental requirements.
• Subdivided into four groups based on phenotypic and genetic properties, representing four species.
• Historically classified into C. botulinum.

Biology and Virulence

• Seven distinct botulinum toxins (A to G) are produced.
• Human disease commonly caused by types A and B.
• Types E and F are also associated with human disease.
• Botulinum toxin, a neurotoxin at the neuromuscular junction, prevents acetylcholine release.
• Blocks neurotransmission at peripheral cholinergic synapses, leading to facial paralysis.
• It acts as an endopeptidase, an enzyme.
• Regeneration of nerve endings is needed to recover function.

Epidemiology

• C. botulinum spores exist worldwide in soil.
• There are relatively few cases of botulism in the United States.
• Botulism is more prevalent in developing countries.
• Infant botulism is more common than other forms in the United States.

Diseases

• Foodborne botulism is characterized by blurred vision, dry mouth, constipation, and abdominal pain.
• It leads to progressive weakness of peripheral muscles and flaccid paralysis.
• Infant botulism has initial nonspecific symptoms, such as constipation, a weak cry, and failure to thrive.
• Infant botulism can progress to flaccid paralysis and respiratory arrest.
• Wound botulism has a similar clinical presentation to foodborne botulism.
• Wound botulism has a longer incubation period and fewer gastrointestinal symptoms.
• Inhalation botulism causes rapid onset of flaccid paralysis and pulmonary failure, along with high mortality.
• This results from inhalation exposure to botulinum toxin.

Diagnosis

• Toxin activity in implicated food or the patient's serum, feces, or gastric fluid confirms foodborne botulism.
• Infant botulism is confirmed the toxin is in the infant’s feces or serum, or the organism cultured from feces.
• Detection of toxin in the patient’s serum or wound confirms wound botulism.
• The organism can also be cultured from the wound.

Treatment, Prevention and Control

• Treatment includes administering metronidazole or penicillin, trivalent botulinum antitoxin, and ventilatory support.
• Trivalent botulinum antitoxin (against toxins A, B, and E) deactivates unbound circulating toxin, reducing mortality.
• Antibodies and memory cells are not produced after the disease.
• Future infections will still be as severe.
• Maintaining food at an acidic pH, high sugar content or storing foods at 4°C or colder prevents spore germination.
• Toxin is heat labile; heating for 10 minutes at 60°C to 100°C destroys it.
• Infant botulism is associated with ingesting contaminated soil or food, like honey.

Clostridium difficile

• Is a large, anaerobic rod known for abundant spore formation, rapid growth in culture, and volatile fatty acids.
• Forms both in-vivo and in-vitro.
• Easily culturable in the laboratory, vegetative cells die rapidly when exposed to oxygen.
• Produces a barnyard smell in culture and two toxins.
• One enterotoxin (enterotoxin A) attracts neutrophils and stimulates their release of cytokines.
• One cytotoxin (cytotoxin B) increases intestinal wall permeability, causing diarrhea.
• A new virulent strain exists caused by a mutation in genes that makes the strain more functional.
• The binary toxin is a useful marker for the strain and adherence to host cells.
• Spore formation enables organism to persist in hospital setting and resist decontamination.
• Antibiotic resistance (clindamycin, cephalosporins, and fluoroquinolones) allows C.difficile to overgrow intestinal bacteria.
• Overgrowth is likely to occur in patients exposed to these antibiotics

Epidemiology

• Colonizes intestines of a small proportion, less than 5%, of healthy individuals.
• Exposure to antibiotics associates with overgrowth of C. difficile and subsequent endogenous infection.
• Spores can be detected in hospital rooms, especially around beds and bathrooms, and can be an exogenous source.

Diseases

• Acute diarrhea generally develops 5-10 days after starting antibiotics and may be brief or protracted.
• Antibiotics include clindamycin, penicillin, cephalosporins, and fluoroquinolones.
• Pseudomembranous colitis is the most severe form of C.difficile disease.
• Symptoms of Pseudomembranous colitis include profuse diarrhea, abdominal cramping, fever, and whitish plaques.
• Whitish plaques form over intact colonic tissue.
• Toxin A (enterotoxin) and toxin B (cytotoxin) are virulence factors.
• Clinical features include diarrhea, bowel inflammation, necrotic bowel, WBCs, and fibrin forming a pseudomembrane.
• Lab diagnosis usually detects C. difficile via EIA or PCR.

Diagnosis

• C. difficile disease is confirmed via detecting cytotoxin or enterotoxin or the toxin genes in the patient's feces.

Treatment, Prevention and Control

• The implicated antibiotic should be discontinued.
• Use metronidazole or vancomycin if there is severe disease.
• Fecal transplants from a healthy donor "repoopulate" to treat recurrent diseases.
• C. difficile cannot become established in the presence of healthy enterobacteria during relapse.
• Relapse is common because antibiotics do not kill spores; a second course may be successful.
• Thoroughly clean the hospital room after the infected patient is discharged because spores are hard to eliminate.

Clostridium perfringens

• Consists of large Gram +, spore-positive rods that rarely are observed.
• Many decolorized rods appear gram-negative, and there is an absence of spores and blood cells.
• Distinct colony morphology and rapid growth.
• Produces toxins and enzymes that lyse blood cells and destroy tissues.
• This results in overwhelming sepsis, massive hemolysis, and myonecrosis.
• Flat spreading colonies with a zone of complete hemolysis is theta toxin and a wider zone of partial hemolysis for alpha toxin.
• Beta toxin causes intestinal stasis, loss of mucosa, and formation of necrotic lesions leading to necrotizing enteritis.
• Epsilon toxin is a protoxin activated by trypsin and increases permeability of the gastrointestinal wall.
• Iota toxin produced by type E C. perfringens increases vascular permeability.
• Phospholipase C Produced by all five types causes hemolysis via lysing erythrocytes.
• Type A strains produce enterotoxin whose activity is enhanced by trypsin exposure.
• The heat-sensitive enterotoxin binds to receptors on brush border of the epithelium of the small intestine.
• Binding leads to loss of fluids and ions (watery diarrhea) and altered membrane permeability.

Epidemiology

• C. perfringens is ubiquitous and present in soil, water, intestinal tract of humans and animals.
• Type A strains responsible for most human infections.

Diseases

• Food poisoning is associated with contaminated meat products held between temperatures of 5°C and 60°C.
• This encourages high organism growth.
• There is a short incubation period.
• Rapid onset of abdominal cramps and watery diarrhea.
• Commonly there is no fever, nausea, or vomiting and a short duration of the illness that is self-limited.
• Type C strain beta-toxin can cause acute necrotizing destruction of jejunum known as "pigbel".
• Pigbel presents with abdominal pain, vomiting, bloody diarrhea, and peritonitis; mortality approaches 50%.
• Soft-tissue infections are typically associated with bacterial contamination of wounds or localized trauma.
• Cellulitis presents as localized edema and erythema with gas formation in the soft tissue and is generally nonpainful.
• Suppurative myositis involves pus accumulation in the muscle planes without muscle necrosis or systemic symptoms.
• Myonecrosis results in rapid destruction of muscle tissue, systemic spread, and high mortality.
• There is painful muscle destruction.

Cellulitis & Gangrene

• Clostridia introduced into tissue during surgery or traumatic injury.
• Followed by skin discoloration, bullae formation, and necrosis, with serosanguineous exudate but no muscle necrosis.
• Gas gangrene is commonly caused by C. perfringens.
• It showcases the full virulence with intense plain developing within a week of trauma or surgery.
• The bacterial agent causes muscle necrosis, shock, renal failure, and death within days of initial onset.
• The wounds become contaminated and the muscle becomes necrotic.

Diagnosis

• Diagnosis relies on Gram-stained tissue specimens with large Gram + rods & rapid culture growth
• Tissue specimens have Gram + rods.

Treatment, Prevention & Control

• Rapid and aggressive is needed to prevent death if a serious infection occurs.
• Proper cleansing and care for wounds also prevents infection, as well as use of prophylactic antibiotics.

Clostridium tetani

• Spores are larger than diameter of cells giving drumstick appearance.
• Difficult to detect because organism is extremely oxygen-sensitive.
• When seen in culture, growth appears as film, not discrete colonies.
• These bacteria are proteolytic but cannot ferment carbohydrates.
• The bacteria are proteolytic and unable to ferment carbohydrates.
• The virulence factor, tetanospasmin, is a heat-labile neurotoxin blocking neurotransmitter release for inhibitory synapses.

Epidemiology

• Ubiquitous spores are present in soils.
• The spores colonize gastrointestinal tract of humans and animals.
• Exposure is common, is is uncommon due to vaccines, medical care.
• Greatest risk is in people with inadequate vaccine-induced immunity.
• This disease does not induce immunity.

Diseases

• Disease is characterized by muscle spasms and autonomic nervous system involvement.
• Generalized tetanus is the more common form that involves masseter muscles, sardonic smile (risus sardonicus), and lockjaw.
• Early signs include drooling, sweating, irritability, consistently recurring back spasms (opisthotonus).
• Generalized musculature spasms and autonomic nervous system involvement manifest as cardiac arrhythmias.
• Other symptoms may include changes in blood pressure, profound sweating, and dehydration.
• Localized tetanus presents musculature spasms restricted to the localized area of the primary infection.
• Neonatal tetanus develops from neonatal infection of the umbilical stump.
• Cephalic tetanus' primary infection site is in the head, and prognosis is very poor

Tetanus

• Clostridium tetani is the causative agent.
• The disease occurs when contaminated materials or surgery introduce the agent into the skin.
• Prolonged muscle spasms in the CNS are caused by tetanospasmin.
• The clinical presentation includes muscle spasm, hyperexcitability, and odd facial expressions.
• Lab diagnosis identifies GPB with oval, terminal or subterminal spores with a "drumstick" or "tennis racket."
• Prevention involves immunization.

Diagnosis

• Diagnosis is based on clinical presentation and not laboratory tests.
• Testing via microscopy, culture, tetanus, toxin, or antibodies is typically insensitive.

Treatment, Prevention and Control

• Treatment requires debridement, antibiotic therapy by penicillin, metronidazole and passive immunization.
• Passive immunization with antitoxin-globulin, and vaccination with tetanus toxoid is also necessary.
• Prevention includes 3 doses of initial tetanus, followed by booster doses every 10 years.

Bacillus

• Genera have 50+ species.
• Spore formers resistant to radiation, heat, disinfections, and desiccation.
• Habitat is widespread: thermal springs, fresh and salt water, soil, and on plants.
• Aerobic organisms distinct from Clostridium are mostly contaminants on clinical samples except Bacillus anthracis.

Spores

• Resistant to environmental changes and dry heat and some chemical disinfectants for moderate periods.
• They can persist for years in dry earth.
• These are produced when bacteria get stressed (induced), by drying/desiccation and temperature extremes.
• Provides microorganisms an advantage in survival.
• Gram stain shows spores as clear areas within the bacterial cell.
• Bacillus produces free spores without vegetative cells.

Significant Bacillus Species

• Bacillus anthracis is responsible for anthrax, primarily a disease in livestock/herbivores.
• Humans acquire infection incidentally by wound contamination, ingestion, and inhalation of spores through the mouth.
• Gasterointestional tracks.
• Bacillus cereus causes gastroenteritis, eye infection, catheter-related sepsis, and pneumonia as an opportunistic pathogen.
• Bacillus subtilis is a common lab contaminant used in sterility testing for endospore resistance.

Bacillus anthracis Infection

• Entry usually through spores though gastrointestinal tracts.
• Lesions have malignant pustule (black eschar) in center, thus are called "rag-pickers disease".
• Organisms gain access through cuts, which will initially resemble insect bites.
• Fever, malaise, and headaches may be early signs.
• Inhalation anthrax (Wool sorter's disease) stems from acquired spores in the respiratory tract.
• This results in respiratory distress, or death.
• Gastrointestinal anthrax acquired by contaminated raw meat is rare but abdominal pain, vomiting, are clinical signs.
• With Injection, spores results in gelatinous edema and congestion causing Outbreaks from the injection site.
• Bacilli spread via lymphatics where they multiply.

Laboratory Diagnosis

• Rules out Bacillus anthracis.
• If Bacillus anthracis is suspected, one must work under safety.
• Other Bacillus identified up to genus only.
• Most species beta-hemolytic on the sheep blood , except B.anthracis.
• Fast growers with varying colony characteristics.

trigger words

• Is spore-former, capsule-formers, edema toxin, lethal toxin, anthrax, bioterrorism.

Biology

• Non-motile, nonhemolytic.
• 3 virulents
  • Protective
  • Edema
  • Lethal
  • individually, they are not toxic but becomes
  • inhibits phagocytosis

treatment

• ciprofloxacin or doxycycline (prevent bacteria), rifampin, vanco, penillican clindaycin or clarithromyicn.
• Vaccine herd or at risk human only
• less toxic vacccine

Microscopic

• In wounds or with lymph node of blood samples
• Appears as long chain strands with spore visible in the culture
• capsule in produced in In-vivo
• SBA = Colony large, but not pigmented

Bacillus cereus

• • is spore-former, enteroxin and causes eye infections.
• important pathogen, it does cause ( gastroenteritis, ocular infections, and catheter-related sepsis.)

Virulence

• Causes infections, such as pneumonia.
• tissue destruction, Heat-labile, cereolysin
• Present in soils
• caused by rice, meat , iv injections.
• Ocular infection = rapid/ progress
• Severe pulmonrary

Food poisoning

• true infection b/c contaminated

Bacillus Subtillus

• Spore producing is for quality production.

Culture

• Anaerobes grow to survive b/c molecular is not presernt
• Most strict cant survive if present.

Incubation

• is usually required 1 to 6 days.
• Use the system

Antimocorbial

• is not routinely performed.
• Chlor.
• Perform lactmase for Gram negative.

Overview of Clostridium botulinum, the cause of botulism. Details its biology, virulence factors, including the seven distinct botulinum toxins (A to G) and their mechanism of action at the neuromuscular junction.