Lecture 1: Clostridium

clostridium: shape

rods (bacilli)

clostridium: gram class

gram-positive rods

clostridium: oxygen classification

anaerobic

clostridium: do they form spores

yes

clostridium: where are they present

Ubiquitously present in soil, water, and sewage and as a normal microbial population in the gastrointestinal (GI) tracts of animals and humans

clostridium: most members of the class are

harmless saprophytes

3 attributes that allow clostridium to cause disease

ability to form spores

ability to grow rapidly in oxygen deprived conditions

ability to produce toxins

Clostridium perfringens: shape, gram, spore or no spores

rectangular, gram-positive rod with spores

C. perfringens: hemolysis pattern

characteristic inner and outer zones of hemolysis while growing on blood agar

C. perfringens: toxins produced

one or more “major lethal” toxins (alpha, beta, epsilon, iota toxins) and enterotoxin

C. perfringens: common habitats

the intestinal tract of humans and animals and is widely distributed in nature, particularly in soil and water contaminated with feces

C. perfringens: spores

Spores form under adverse environmental conditions and survive for prolonged periods

C. perfringens: diseases caused

soft-tissue infections, food poisoning, necrotizing enteritis, and septicemia

C. perfringens: inner clear hemolytic zone

Complete destruction of red blood cells (β-hemolysis)

Caused primarily by theta toxin (perfringolysin O)

C. perfringens: outer zone of hemolysis

Partial destruction of red blood cells

Caused primarily by alpha toxin (phospholipase C)

C. perfringens: alpha toxin

lyseerythrocytes, platelets, leukocytes, and endothelial cells, resulting in hemolysis, increased vascular permeability and bleeding

C. perfringens: beta toxin

responsible for intestinal stasis, loss of mucosa with formation of necrotic lesions and necrotizing enteritis (inflammation of small intestine)

C. perfringens: epsilon toxin

increases the vascular permeability of the GI wall

C. perfringens: iota toxin

has necrotic activity towards endothelial cells and increases vascular permeability

C. perfringens: theta toxin

cholesterol-binding cytolysin that lyse the target cell and plays a key role in the tissue destruction and vascular injury

C. perfringens: enterotoxin

binds to receptors on the brush border membrane of the small intestine and leads to pore formation, altered membrane permeability, and loss of fluids and ions

C. perfringens: soft-tissue infections caused

cellulitis, fasciitis, and myonecrosis with gas formation in the soft tissue (gas gangrene)

Clostridial myonecrosis

Life-threatening disease that illustrates the full virulence potential of histotoxic clostridia

The onset of disease, characterized by intense pain, generally develops within a week after clostridia are introduced deep into tissue by trauma or surgery

Clostridial myonecrosis: why does it go so deep into tissue

it is anaerobic

Clostridial myonecrosis: what microbe is it caused by

C. perfringens

Clostridial myonecrosis: gas gangrene

Gas found in the tissue is due to metabolic activity of the rapidly dividing bacteria (hydrogen and carbon dioxide)

Clostridial myonecrosis: gram stain

Gram stain of tissue collected from a patient with C. perfringens myonecrosis will reveal abundant rectangular gram-positive rods with the absence of inflammatory cells (resulting from lysis by clostridial toxins)

Clostridial myonecrosis: what do toxins cause

extensive hemolysis and bleeding

Clostridial myonecrosis: 4 steps of treatment

1. Immediate surgery (debridement) to remove all dead and infected tissue

2. High-dose IV antibiotics (commonly penicillin plus clindamycin)

3. Hyperbaric oxygen therapy in selected cases

4. Amputation

Clostridial myonecrosis: Hyperbaric oxygen therapy

creates an environment too oxygen-rich for anaerobic bacteria; directly inhibits production of C. perfingens alpha-toxin

promotes tissue repair and halts rapid disease spread

Clostridial myonecrosis: 4 cases where amputation may be required

The infection has extensively destroyed muscle and soft tissue

The infection continues to spread despite aggressive debridement

Blood supply to the limb is severely compromised

Limb salvage would endanger the patient's life

C. perfringens food poisoning

(1) a short incubation period (8–12 hours); (2) abdominal cramps and watery diarrhea but no fever, nausea, or vomiting; (3) a clinical course lasting less than 24 hours

C. perfringens food poisoning: disease results from ingestion of

Disease results from ingestion of meat products contaminated with large numbers of enterotoxin-producing C. perfringens

C. perfringens food poisoning: what temperature allows spores to germinate

Holding contaminated foods at temperatures less than 60°C allows spores that survived the cooking process to germinate and multiply to high numbers

C. perfringens food poisoning: how to prevent bacterial growth

Rapid refrigeration of food after preparation prevents this bacterial growth. Alternatively, reheating the food to 74°C can destroy the heat-labile enterotoxin

C. perfringens: Necrotizing enteritis

rare necrotizing process in the jejunum characterized by acute abdominal pain, vomiting, bloody diarrhea, ulceration of the small intestine, and perforation of the intestinal wall, leading to peritonitis and shock

Necrotizing enteritis: what toxin causes disease

Beta toxin produced by C. perfringens is responsible

Clostridium tetani: motility, spore, shape

motile, spore-forming rod

Clostridium tetani: spores

round, terminal spores that give it the appearance of a drumstick

Clostridium tetani: protein and carbohydrate breakdown

proteolytic and are unable to ferment carbohydrates

Clostridium tetani: toxins produced

two toxins, an oxygen-labile hemolysin (tetanolysin) and a plasmid-encoded, heat-labile neurotoxin (tetanospasmin)

tetanospasmin: when is it released and what is it responsible for

released when the cell is lysed, and responsible for the clinical manifestations of tetanus

tetanospasmin: structure

an A-B toxin with a disulfide bond holding the two chains together

tetanospasmin: what does it inactivate

It inactivates proteins that regulate release of the inhibitory neurotransmitters glycine and gamma-aminobutyric acid (GABA)

tetanospasmin: what does its release lead to

unregulated excitatory synaptic activity in the motor neurons, resulting in spastic paralysis

tetanospasmin: role of A and B unit

A: activating unit

B: binding unit

tetanospasmin: steps of action

1. B-unit binds and toxin is taken up at the neuromuscular junction

2. Retrograde transport to CNS and entry into inhibitory interneurons

3. A unit is released and cleaves SNARE proteins, blocking release of inhibitory neurotransmitters (GABA or glycine)

most common form of tetanus

generalized tetanus

generalized tetanus: muscles involves

Involvement of the masseter muscles (trismus or lockjaw) is the presenting sign in most patients

generalized tetanus: sardonic smile

results from the sustained contraction of the facial muscles is known as risus sardonicus

early signs of generalized tetanus

drooling, sweating, irritability, and persistent back spasms (opisthotonos), with more severe symptoms including cardiac arrhythmias, fluctuations in blood pressure, profound sweating, and dehydration

localized tetanus

the disease remains confined to the musculature at the site of primary infection

cephalic tetanus

locailzed form of generalized tetanus; primary site of infection is the head

why is localized tetanus rare in the US

the high incidence of vaccine-induced immunity

neonatal tetanus

typically associated with an initial infection of the umbilical stump that progresses to become generalized

Mortality in infants exceeds 90%, and developmental defects are present in survivors; almost exclusively a disease in developing countries

Tetanus management

The primary challenge is preventing violent, agonizing muscle spasms.

Patients are kept in dark, quiet rooms to minimize sensory triggers.

Given high doses of continuous sedatives and muscle relaxants

Tetanus management: medication in severe cases

given full neuromuscular blocking drugs

Clostridium botulinum: nutrition requirement, spore, oxygen classification, shape

fastidious, spore-forming, anaerobic rods

Clostridium botulinum: common locations

soil and water

Clostridium botulinum: how many toxins

Eight antigenically distinct botulinum toxins (A, B, C1, C2, D, E, F, and G) have been described

Clostridium botulinum: what toxins are associated with human disease

A, B, E, and F

Botulinum toxin: structure

A-B toxin consisting of a small subunit (light, or A chain) and a large, nontoxic subunit (B, or heavy chain)

Botulinum toxin: carboxyl-terminal portion of heavy chain

binds specific glycoproteins (different from those targeted by tetanospasmin) on the surface of excitatory neurons and stimulates endocytosis of the toxin molecule

botulinum toxin: how is N-terminal of heavy chain stimulated, and what effect does it have

Acidification of the endosome stimulates N-terminal, heavy chain–mediated release of the light chain that then inactivates the proteins that regulate release of acetylcholine, blocking neurotransmission

botulinum toxin: effect of release and acetylcholine blocking

Because acetylcholine is required for excitation of muscle, the resulting clinical presentation of botulism is a flaccid paralysis (floppy limbs, muscle weakness, difficulty breathing and eating); Life-threatening

Both, tetanus and botulism requires

regeneration of nerve endings

4 forms of botulism

classic, infant, wound, inhalation

Classic or foodborne botulism

Associated with consumption of home-canned foods. The food may not appear spoiled, but even a small taste can cause full-blown clinical disease. Avoid eating canned foods that are expired, damaged or look bloated. It is heat-labile, meaning high temperatures can destroy it

Infant botulism

More common and has been associated with consumption of foods (e.g., honey, infant milk powder) contaminated with botulinum spores and ingestion of spore-contaminated soil and dust

Wound botulism

Seen in black tar heroin users through contaminated needles, or deep puncture wounds, gunshot wounds, surgery wounds

Inhalation botulism

Rare man-made form and is a major concern in this era of bioterrorism. Botulinum toxin has been concentrated for purposes of aerosolization as a biological weapon. When administered in this manner, inhalation disease has a rapid onset and potentially high mortality

The clinical diagnosis of foodborne botulism is confirmed if

toxin activity is demonstrated in the implicated food or in the patient’s serum, feces, or gastric fluid

Isolation of C. botulinum from specimens contaminated with other organisms can be improved by

heating the specimen for 10 minutes at 80°C to kill all non–spore-forming bacteria

botulism: treatment

adequate ventilatory support and use of botulinum antitoxin to inactivate unbound toxin circulating in the bloodstream. Ventilatory support is extremely important in reducing mortality

botulism: when are antibiotics indicated

may be helpful in wound botulism but is not recommended for other forms of botulism because the lysis of the bacteria may release more toxin

botulism: how to prevent

by preventing spore germination (by maintaining the food in an acid pH or storage at 4°C or colder) or destroying the preformed toxin (all botulinum toxins are inactivated by heating at 60°C–100°C for 10 minutes)

Clostridium difficile: oxygen classification, spores, shape

anaerobic spore-forming rod

Clostridium difficile: macromolecule produced

produces a variety of volatile fatty acids that produce a characteristic “barnyard/horse manure” smell in culture

Clostridium difficile: toxins produced

an enterotoxin (toxin A) and a cytotoxin (toxin B)

Clostridium difficile: toxin A

chemotactic for neutrophils, stimulating the infiltration of neutrophils into the ileum with release of cytokines resulting in inflammation

Also produces a cytopathic effect, resulting in disruption of the tight cell-to-cell junction, increased permeability of the intestinal wall, and subsequent diarrhea

Clostridium difficile: toxin B

The cytotoxin causes actin to depolymerize, with resultant destruction of the cellular cytoskeleton

Clostridium difficile: significance of bacterial surface layer proteins

important for the binding of C. difficile to the intestinal epithelium, leading to localized production of toxins and subsequent tissue damage

Clostridium difficile: typical location in flora

C. difficile is part of the normal intestinal flora in a small number of healthy people and hospitalized patients

Clostridium difficile: when does disease manifest

Develops in people taking antibiotics since the drugs alter the normal enteric flora, either permitting overgrowth of these relatively resistant organisms or making the patient more susceptible to exogenous acquisition of C. difficile

Clostridium difficile: in disease what does the bacteria penetrate

the organisms proliferate in the colon and produce their toxins

Toxin-producing C. difficile is the most common cause of

antibiotic-associated GI diseases ranging from a self-limited diarrhea to severe, life-threatening pseudomembranous colitis

Pseudomembranous colitis: diagnosis

confirmed by demonstration of the enterotoxin or cytotoxin in a stool specimen from a patient with compatible clinical symptoms

Pseudomembranous colitis: symptoms

Watery diarrhea, severe abdominal pain, fever, nausea, and blood/pus in the stool

Pseudomembranous colitis: treatment

Discontinuing the antibiotic, medications like vancomycin or fidaxomicin to clear C. difficile infection, IV fluids and electrolytes, Fecal microbiota transplant

Pseudomembranous colitis

Advanced inflammation of colon. Results in formation of pseudomembranes (mucus, debris from dead cells, clotted blood, immune cells)