Chapter 6: Gram-positive Bacteria: Spore-forming rods: Bacillus and Clostridium Flashcards
What is the Gram stain and morphology of Bacillus?
Gram-positive
spore-forming
rods
What are the two Gram-positive spore-forming rods?
Bacillus
Clostridium
What is the Gram stain and morphology of Clostridium?
Gram-positive spore-forming rods
What type of metabolism does Bacillus exhibit?
aerobic
What type of metabolism does Clostridium exhibit?
anaerobic
like in a closet
How do Bacillus and Clostridium differ biochemically to oxygen?
Bacillus is aerobic.
Clostridium is anaerobic.
How do Bacillus and Clostridium cause disease?
By the release of potent exotoxins
What bacterium causes the disease anthrax?
Bacillus anthracis
What are the two pathogenic species of Gram-positive aerobic spore-forming rods?
Bacillus anthracis
Bacillus cereus
Bacillus anthracis - Gram stain
Gram-positive
Bacillus anthracis - morphology
rods
Bacillus anthracis - spores-forming?
yes
Bacillus anthracis - motility?
non-motile
Bacillus anthracis - capsule?
encapsulated (poly-D-glutamic acid; only one with protein capsule)
Bacillus anthracis - metabolism?
aerobic
What is the only bacterium with a capsule composed of protein?
Bacillus anthracis
Bacillus anthracis has a capsule composed of what?
protein - poly-D glutamic acid
How does the Bacillus anthracis capsule act as a virulence factor?
It prevents phagocytosis by macrophages.
How is Bacillus anthracis transmitted? (3)
skin contact
inhalation
ingestion of spores (from contaminated meat)
What population is particularly vulnerable to Bacillus anthracis infection?
individuals exposed to infected herbivores (cows and sheep) or soil, their skins (hides or wool), or their carcasses
Can Bacillus anthracis be transmitted from human to human?
No, it has never been reported. Infection only occurs when humans are exposed to the spores during direct contact with infected animals or soil.
Where are Bacillus anthracis spores present?
in dry soil, and in the GI tract of infected animals (zoonotic herbivores: sheep, goats, cattle)
What are the 3 forms of anthrax? (related to modes of transmission)
1) cutaneous anthrax
2) respiratory anthrax
3) GI anthrax
How does cutaneous anthrax present?
painless necrotic pustules - round black lesion (boil-like): ulcer with black eschar, often with a rim of edema of surrounding tissue.
Uncommonly progresses to bacteremia and death
How does pulmonary (inhalation) anthrax present?
flu-like symptoms (non-specific myalgias, fever, chest pain, and cough) that rapidly progress to fever, pulmonary hemorrhage, mediastinitis (leading to widening of the mediastinum), leading to bacteremia/shock in a fatal fulminant phase.
What is woolsorter’s disease?
Pulmonary anthrax resulting from inhalation of spores from contaminated wool.
How does inhalation of Bacillus anthracis spores lead to mediastinitis?
The spores are taken up by the macrophages in the lungs and transported to the hilar and mediastinal lymph nodes where they germinate. Mediastinal hemorrhage occurs, resulting in mediastinal widening and pleural effusions.
What is the most common route of entry of Bacillus anthracis?
cutaneous
What is the rarest route of entry of Bacillus anthracis?
GI
How does GI anthrax present?
vomiting, abdominal pain, and dysentery (bloody diarrhea), resulting from the formation of necrotic lesion within the intestine by the exotoxin
Aside from the capsule, what is the major virulence factor of Bacillus anthracis?
exotoxin
The protein components of Bacillus anthracis exotoxin are encoded on what plasmid?
pXO1
What is pXO1?
plasmid that encodes Bacillus anthracis exotoxin
What 3 proteins does pXO1 encode?
1) Protective antigen (PA)
2) Edema factor (EF)
3) Lethal factor (LF)
Protective antigen (PA), Edema factor (EF), Lethal factor (LF) are components of…?
Bacillus anthracis exotoxin (or anthrax toxin)
How does the Bacillus anthracis spore become activated?
Only when introduced into the host. Germination and expression of virulence factors (on plasmids pXO1 and pXO2) is regulated by an increase in temperature to 37deg, CO2 concentration, and serum proteins.
What 3 proteins can comprise anthrax exotoxin?
1) Protective antigen (PA)
2) Edema factor (EF)
3) Lethal factor (LF)
Are the 3 individual protein components of Bacillus anthracis exotoxin toxic individually? What combinations are toxic?
No, they are individually nontoxic. Edema toxin (ET) = Protective antigen (PA) + Edema factor (EF) Lethal toxin (LT) = Protective antigen (PA) + Lethal factor (LF)
Which is more dangerous and rapidly fatal, LT or ET?
Lethal toxin (protective antigen PA + lethal factor LF) is more dangerous and rapidly fatal than Edema toxin (protective antigen PA + edema factor EF)
What is protective antigen (PA), a component of Bacillus anthracis exotoxin?
becomes a channel in the mammalian plasma membrane, promoting entry of edema factor (EF) or lethal factor (LF) into the cytosol of phagocytic cells
What is edema factor (EF), a component of Bacillus anthracis exotoxin?
a calmodulin-dependent adenylate cyclase that increases cAMP, which impairs neutrophil function and causes massive edema and disruption of innate immunity.
What component of Bacillus anthracis exotoxin is a calmodulin-dependent adenylate cyclase?
Edema factor (EF)
What is lethal factor (LF), a component of Bacillus anthracis exotoxin?
a zinc metalloprotease that inactivates MAPK (mitogen-activated protein kinase), which stimulates the macrophage to release TNFa and IL1b, which lead to rapid cell death.
Which component of Bacillus anthracis exotoxin is a zinc metalloprotease that inactivates MAPK?
lethal factor (LF)
What component of Bacillus anthracis exotoxin becomes a channel in the mammalian plasma membrane, promoting entry of edema factor (EF) or lethal factor (LF) into the cytosol of phagocytic cells?
protective antigen (PA)
What is pXO2?
plasmid in Bacillus anthracis that encodes genes necessary for the synthesis of the poly-D-glutamic acid capsule.
Virulence of Bacillus anthracis depends on acquiring…?
2 plasmids: pXO1 (exotoxins) and pXO2 (protein capsule)
What proteins does pXO2 plasmid encode?
enzymes required for the synthesis of Bacillus anthracis poly-D-glutamic acid capsule; to prevent phagocytosis of the bacterium.
Proteins required for the synthesis of Bacillus anthracis capsule are encoded by what plasmid?
pXO2
What is the treatment for systemic Bacillus anthracis infection? (2)
ciprofloxacin
doxycycline
(rapid identification and prompt treatment are critical to prevent mortality from anthrax)
What is the treatment for use in inhalational anthrax?
Raxibacumab (human monoclonal antibody), in combination with antibacterial drugs
The human vaccine for anthrax is composed of what?
protective antigen (PA)
The animal vaccine for anthrax is composed of what?
live cultures attenuated by loss of antiphagocytic protein capsule (too dangerous for human use)
Which high-risk individuals should receive the anthrax vaccine?
high risk activities (petting goats or cows in countries where this disease is rampant), and military personnel
Bacillus cereus - Gram stain
Gram-positive
Bacillus cereus - morphology
rods
Bacillus cereus - spore-forming?
yes
Bacillus cereus - motility?
motile
Bacillus cereus - metabolism
aerobic
Bacillus cereus - capsule?
non-encapsulated (unlike B. anthracis)
How does Bacillus cereus infection present?
as food poisoning: nausea, vomiting, diarrhea
How does Bacillus cereus associated food poisoning occur?
B. cereus deposits its spores in food, which then survive the initial cooking process. The bacteria then germinate in the warm food and begin releasing their enterotoxin.
Reheated rice syndrome is associated with what bacterium?
Bacillus cereus
How many types of enterotoxin does Bacillus cereus secrete? What are they?
1) heat-labile toxin (diarrheal type)
2) heat-stable toxin (emetic type)
Describe the clinical presentation of the emetic type of Bacillus cereus infection, including time course.
short incubation period 1-5 hours, severe nausea and vomiting (no or limited diarrhea)
(similar to Staphylococcus aureus food poisoning)
What toxin is associated with the emetic type of Bacillus cereus infection?
heat-stable toxin (cereulide), in rice or pasta
Describe the clinical presentation of the diarrheal type of Bacillus cereus infection, including time course.
watery, non-bloody diarrhea; nausea and abdominal pain. 8-18 hours.
What toxin is associated with the diarrheal type of Bacillus cereus infection?
heat-labile toxin
Is Bacillus cereus infection responsive to penicillin or other antibiotic treatment?
No, because the food poisoning is caused by the pre-formed enterotoxin
Clostridium botulinum - Gram stain?
Gram-positive
Clostridium botulinum - morphology?
rods
Clostridium botulinum - metabolism?
Anaerobic
like in a closet
Clostridium botulinum - spore forming?
yes
Clostridium botulinum - motility?
Motile: flagella (so H-antigen positive)
Where are Clostridium botulinum spores present? (4)
1) soil
2) stored vegetables: home-canned or zip-lock storage bags
3) smoked fish
4) fresh honey: associated with infant botulism
What disease does infection with Clostridium botulinum cause? What are the clinical symptoms (4)?
Adult and infant botulism resulting from rapidly fatal food poisoning.
1) Diplopia (double vision) or blurry vision
2) Dysphagia (difficulty swallowing) or dry mouth
3) Flaccid muscle paralysis (floppy baby syndrome)
4) Respiratory paralysis and death
Infection with what organism causes the following symptoms:
1) Bilateral cranial nerve palsies: Diplopia (double vision) or blurry vision
2) Bilateral cranial nerve palsies: Dysphagia (difficulty swallowing) or dry mouth
3) Flaccid muscle paralysis
4) Respiratory paralysis and death
Clostridium botulinum
By what mechanism does Clostridium botulinum toxin exert its effects?
Botulinum neurotoxin is a protease that cleaves synaptobrevin fusion proteins, blocking the release of acetylcholine from presynaptic nerve terminals in the autonomic nervous system and motor endplates, causing flaccid muscle paralysis.
How is adult botulism caused?
Ingestion of preformed heat-labile toxin:
C. botulinum spores float in the air and can land on food. If food is cooked thoroughly, spores will die. If not cooked thoroughly and then placed into an anaerobic environment (such as a can or ziplock bag (smoked fish or home-canned vegetables)), C. botulinum matures and synthesizes neurotoxin. Ingesting contents of the jar will be ingesting the potent neurotoxin.
Spores are unable to germinate in the adult GI tract, but can in the infant GI tract.
What microbe is associated with ingestion of smoked fish or home-canned/ziplocked foods in adults?
Clostridium botulinum
BOTulinum is from bad BOTtles of food and honey.
What microbe is associated with ingestion of honey in infants?
Clostridium botulinum, causing floppy baby syndrome.
BOTulinum is from bad BOTtles of food and honey.
How is infant botulism caused?
Infants ingest food contaminated with Clostridium botulinum spores, such as in fresh honey. The spores germinate and colonize in the infant’s GI tract, and from this location the botulinum neurotoxin is released, causing floppy baby syndrome.
What microbe causes floppy baby syndrome? What is the mode of transmission?
Infants ingest food contaminated with Clostridium botulinum spores, such as in fresh honey. The spores germinate and colonize in the infant’s GI tract, and from this location the botulinum neurotoxin is released.
Describe the clinical symptoms of infant botulism.
(floppy baby syndrome caused by ingestion of Clostridium botulinum spores)
- constipation for 2-3 days
- followed by difficulty swallowing and muscle weakness (flaccid paralysis)
How does the mode of transmission of Clostridium botulinum differ in adult versus infant botulism?
In adults, caused by ingestion of preformed toxin in smoked fish or canned foods.
In infants, caused by ingestion of spores in contaminated food.
True or false: Adult botulism is associated with fever.
FALSE - they are afebrile.
- prodromal GI symptoms
- bilateral cranial nerve palsies (double or blurry vision, dry mouth or difficulty swallowing)
- flaccid muscle paralysis
- respiratory paralysis and death
What is the treatment for adult and wound botulism?
Botulinum antitoxin (from equine) (also supportive therapy such as incubation and ventilatory assistance)
What is the treatment for infant botulism?
BIG-IV: human botulism immunoglobulin intravenous
True or false: Wound botulism is associated with fever and elevated WBC.
TRUE! (unlike adult botulism)
Otherwise, the clinical symptoms are the same (except for prodromal GI symptoms)
-bilateral cranial nerve palsies (double or blurry vision, dry mouth or difficulty swallowing)
-muscle weakness and flaccid muscle paralysis
-respiratory paralysis and death
Clostridium tetani - Gram stain?
Gram-positive
Clostridium tetani - morphology?
rods, often with an endospore at one end, giving them the appearance of a drumstick
Clostridium tetani - spore-forming?
yes
Clostridium tetani - motility?
Motile: flagella (so H-antigen positive)
Where are Clostridium tetani spores present?
in soil and animal feces
(tetanus classically follows a puncture wound by a rusty nail, but can follow skin trauma by any object contaminated with spores)
How does Clostridium tetani infection occur?
Spores are commonly found in soil and animal feces, are deposited in a puncture wound and can germinate there as long as there is a localized anaerobic environment (necrotic tissue). From this location, C. tetani releases its exotoxin tetanospasmin. Classically associated with rusty nail puncture wound.
Clostridium tetani - metabolism?
anaerobic
like in a closet
What is trismus?
“lockjaw”
associated with tetanus (disease caused by Clostridium tetani)
What is risus sardonicus?
“ironic smile of tetanus” (disease caused by Clostridium tetani)
What is opisothonos?
pronounced arching of the back
associated with tetanus (disease caused by Clostridium tetani infection)
What is the term for lockjaw, contraction of the jaw muscles?
trismus
What is the term for ironic smile of tetanus, contraction of the facial muscles?
risus sardonicus
What is the term for pronounced arching of the back, contraction of back extensor muscles?
opisthotonos
Is there a vaccine for tetanus?
Yes, the DPT vaccine (diphtheria-pertussis-tetanus): a series of shots at 2, 4, 6, and 18 months, followed by a booster before entry into school (4-6 years), and booster shots every 10 years.
What is DTaP?
vaccine against Diphtheria, (Clostridium) Tetanus, and acellular Pertussis
What is the treatment for infection with Clostridium tetani?
Antitoxin: human tetanus immune globulin (pre-formed anti-tetanus antibodies)
(also metronidazole or penicillin, and supportive therapy such as ventilatory assistance)
What Gram-positive microbe looks like a drumstick?
Clostridium tetani
What disease does Clostridium tetani cause? What are the clinical features (4)?
tetanus: sustained muscle contraction
1) spastic paralysis
2) trismus (“lockjaw”)
3) risus sardonicus (“ironic smile of tetanus”)
4) opisthotonos (pronounced arching of the back)
Infection with which microbe is characterized by:
1) spastic paralysis
2) trismus (“lockjaw”)
3) risus sardonicus (“ironic smile of tetanus”)
4) opisthotonos (pronounced arching of the back)
Clostridium tetani
What exotoxin does Clostridium tetani produce?
tetanospasmin
By what mechanism does Clostridium tetani exert its effects?
Tetanospasmin is taken up at the NMJ and transported to the CNS. The toxin is a protease the cleaves synaptobrevin, and acts on the inhibitory Renshaw cell interneurons in the spinal cord, thereby preventing the release of GABA and glycine from Renshaw cells. This inhibition of inhibitory interneurons allows motor neurons to send a high frequency of impulses to muscle cells, resulting in a sustained tetanic contraction.
Clostridium botulinum and Clostridium tetani both produce a toxin (botulinum toxin and tetanospasmin) which is a protease that cleaves synaptobrevin, thereby inhibiting neurotransmitter release, and both can lead to respiratory failure. How do they differ?
Botulinum toxin inhibits acetylcholine release at the NMJ, leading to flaccid paralysis.
Tetanospasmin inhibits glycine and GABA release from Renshaw interneurons in the spinal cord, leading to rigid paralysis.
Clostridium perfringens - Gram stain?
Gram-positive
Clostridium perfringens - morphology?
rods
Clostridium perfringens - metabolism?
anaerobic
like in a closet
Clostridium perfringens - spore-forming?
yes
Clostridium perfringens - motility?
Non-motile (it is the only Clostridium that is non-motile)
What is the only Clostridium species that is non-motile?
Clostridium perfringens
Where are Clostridium perfringens spores present?
Ubiquitous
1) soil
2) GI tract of humans and mammals
What microbe is associated with gas gangrene?
Clostridium perfringens
What are 3 classes of infection with Clostridium perfringens?
1) Cellulitis/wound infection
2) Clostridial myonecrosis (gas gangrene)
3) Diarrheal illlness
Describe the cellulitis associated with Clostridium perfringens infection.
Necrotic skin is exposed to C. perfringens, which grows and damages local tissue. Palpation reveals a moist, spongy, crackling consistency to the skin due to pockets of gas; this is called crepitus.
What is crepitus?
Moist, spongy, crackling consistency to the skin upon palpation, due to pockets of gas (as produced by infection with microbe like Clostridium perfringens)
Describe the myonecrosis associated with Clostridium perfringens infection.
When inoculated with trauma into muscle, it secretes many exotoxins that destroy adjacent muscle. They also release other enzymes that ferment carbohydrates, resulting in gas formation. As the enzymes degrade the muscles, a thin blackish fluid exudes from the skin. Clostridial myonecrosis (gas gangrene) is fatal if untreated.
Describe the diarrheal illness associated with Clostridium perfringens infection.
Spores can germinate in foods such as meats, poultry, and gravy. Ingestion of large amounts of bacteria can lead to enterotoxin production in the gut and subsequent watery diarrhea.
What two main exotoxins does Clostridium perfringens produce?
1) lecithinase (alpha toxin)
2) an enterotoxin
What is the most important and most lethal exotoxin secreted by Clostridium perfringens?
Lecithinase (alpha toxin)
C. perfringens has more than 12 lethal tissue invasive toxins
What microbe produces lecithinase (aka alpha toxin)?
Clostridium perfringens
also Listeria monocytogenes to a lesser extent
What is the mechanism of action of lecithinase (aka alpha toxin)?
It hydrolyzes lecithin, a key component of cell membranes, resulting in cell death. Lecithinase can be directly inoculated into tissue in the event of trauma to the skin or muscle.
How can x-ray or CT be used for diagnosis of Clostridium perfringens?
reveals pockets of gas within the muscles and subcutaneous tissue
What is the treatment for Clostridium perfringens infection (gas gangrene)?
Radical surgery (may require amputation): excision of necrotic tissue, exposure of anaerobic spaces to oxygen
- is responsive to penicillin
- supportive treatment, such as hyperbaric oxygen
Clostridium difficile - Gram stain?
Gram-positive
Clostridium difficile - morphology?
rods
Clostridium difficile - metabolism?
anaerobic
like in a closet
Clostridium difficile - spore-forming?
yes
Clostridium difficile - motility?
motile: flagella (so H-antigen positive)
Where are Clostridium difficile spores found?
GI tract
commonly found in hospitals and nursing homes
What clinical symptoms are associated with Clostridium difficile infection?
1) severe diarrhea
2) abdominal cramping
3) fever
What is the most common cause of nosocomial diarrhea?
Clostridium difficile
What microbe is associated with pseudomembranous enterocolitis?
Clostridium difficile (antibiotic-associated diarrhea)
How does disease arise from Clostridium difficile infection?
Use of broad-spectrum antibiotics (clindamycin and ampicillin) can wipe out part of the normal intestinal floral, allowing the pathogenic C. difficile that is sometimes present to flourish and superinfect the colon. Once C. difficile grows in abundance, it then releases its exotoxins (A & B).
What exotoxins does Clostridium difficile produce? (2)
Toxin A (enterotoxin) Toxin B (cytotoxin)
What is Toxin A, produced by Clostridium difficile?
An enterotoxin which attracts neutrophils, which release cytokines and cause mucosal inflammation and gastrointestinal fluid loss (severe diarrhea).
What is Toxin B, produced by Clostridium difficile?
A cytotoxin, which disrupts the cytoskeleton by depolymerizing actin filaments, causing GI mucosal cell death and eventually pseudo-membranous colitis (diarrhea).
How is Clostridium difficile infection diagnosed?
stool culture followed by PCR detect Toxin A and/or Toxin B genes
(results available within an hour; excellent specificity and sensitivity)
What is the treatment for Clostridium difficile infection? (3)
1) metronidazole (for mild cases) (oral or IV)
2) oral vancomycin (for severe disease or relapse)
3) fecal transplant for recurring cases to prevent relapse
Why is oral vancomycin unique and effective treatment against Clostridium difficile infection?
Vancomycin is not absorbed when taken orally, so it remains in the GI tract and sits at the site of infection.
Is IV vancomycin effective treatment against Clostridium difficile infection? Why or why not?
No! IV vancomycin does not get into the gastrotintestinal tract and is worthless against colitis due to C. difficile!
Should vancomycin to treat Clostridium difficile be given orally or IV?
Oral
Vancomycin is not absorbed when taken orally, and remains in the GI tract at the site of infection. IV vancomycin does not get into the GI tract and is worthless against colitis due to C. difficile
Should metronidazole to treat Clostridium difficile infection be given orally or IV?
Either - metronidazole is well absorbed into the bloodstream and is carried to the site of infection via the colonic blood vessels
