Anaerobes

Question 1

What causes molecular oxygen to be toxic to anaerobic bacteria?

Answer 1

The toxicity of molecular oxygen to microorganisms is largely due to production of hydrogen peroxide and superoxide. In aerobic organisms these by-products are detoxified by catalase (and peroxidases) and superoxide dismutase, respectively. However, these enzymes are typically absent from the strict anaerobes. Most anaerobes that are frequently associated with disease are aerotolerant; the presence of catalase, peroxidase or superoxide dismutase are virulence factors in these strains.

Question 2

What are the two primary divisions of anaerobic bacteria?

Answer 2

i) The anaerobic Gram-negative rods and anaerobic cocci (especially Bacteroides), and
ii) The members of the genus Clostridium (Gram-positive, spore-forming rods)

Question 3

What is the typical pathogenesis of the anaerobic Gram-negative rods and anaerobic cocci?

Answer 3

Members of the normal microbial flora and rarely cause disease without predisposing factors. Disease most often results from AUTOINFECTION wherein the normal flora transgress epithelial barriers through trauma or pathologic conditions, or because of compromised immune defenses. Only a few of the hundreds of species are typically encountered in a pathologic state. Members of the Bacteroides group are the most frequently encountered; Bacteroides fragilis is considered to be the prototypical endogenous anaerobic pathogen.

Question 4

What makes anaerobic infections biphasic?

Answer 4

Anaerobic infections are typically “polymicrobial”, or “mixed infections”, containing both anaerobic and facultative or aerobic organisms. In the early acute stage of infection aerobes and facultative organisms predominate. Their metabolic activity creates an anaerobic microenvironment allowing the subsequent growth of anaerobes. This later stage of infection in which anaerobes predominate is typically characterized by chronic abscess formation. The abscess is conducive to maintaining a low oxygen tension environment and provides protection from host defenses and antimicrobial agents.

Question 5

How are anaerobic infections treated?

Answer 5

Treatment of anaerobic infections frequently requires surgical drainage of abscesses and debridement of devitalized tissue, along with appropriate antimicrobial agents.

Question 6

What are the virulence factors of B. fragilis?

Answer 6

1. Fimbrae (pili) that mediate adherence to epithelial cells.
2. Extracellular enzymes such as phospholipase A, collagenase, heparinase result in tissue damage and contribute to infection.
3. Superoxide dismutase (in conjunction with catalase) enhances the ability of the organism to survive under less than stringent anaerobic conditions in the host.
4. Polysaccharide capsule. This unique capsule is a target the humoral and cellular immunity. The capsule likely contributes to abscess formation, since injected purified capsule can incite abscess formation in the absence of viable organisms.

Question 7

What common virulence factor is missing from B fragilis, and what effect does this have?

Answer 7

B. fragilis LPS lacks the lipid A (endotoxin) moiety. Thus, septic shock is not commonly associated with B. fragilis bacteremias.

Question 8

What are the four most pathogenic species in genus Clostridium, and what are their associated diseases?

Answer 8

C. tetani tetanus
C. botulinum botulism
C. perfringens food poisoning and gas gangrene
C. difficile diarrhea

Question 9

What type of toxin do C. tetani and C. botulinum produce and what effects do these toxins have?

Answer 9

Both are “typical” two subunit AB toxins; one subunit promotes specific host-cell binding and uptake, and the second subunit is a specific protease that mediates the toxic activity. The mechanism of action is cleavage of specific proteins at the synaptic cleft, thereby blocking release of neurotransmitters.

Question 10

How is C. tetani commonly acquired and what is the pathogenesis of its associated disease?

Answer 10

C. tetani spores are usually introduced into wounds by environmental contamination. Locally produced tetanus toxin (tetanospasmin) is transported to the CNS by retrograde axonal transport along peripheral motor neurons.
Toxin binds to gangliosides particularly those found on presynaptic internucial cells of the spinal cord (Renshaw cells). There, the toxin interferes with inhibitory synapses by blocking release of the inhibitory neurotransmitters (GABA, glycine). This results in unopposed motor excitation and generalized spasticity (spastic paralysis). Toxin binding is irreversible; recovery requires formation of new axonal terminals.

Question 11

What are the clinical manifestations of C. tetani?

Answer 11

Incubation period ranges from a few days to 3 weeks. Generalized tetanus is characterized by trismus (lockjaw), neck stiffness, difficulty swallowing, rigidity of abdominal and back muscles and fever. The highest mortality is in newborns where disease progression is rapid.

Question 12

How is tetani treated and prevented?

Answer 12

Treatment of patients with suspected tetanus includes
i) debridement of the wound
ii) treatment with an antimicrobial such as metronidazole
iii) passive immunization with human tetanus immunoglobulin,
iv) vaccination with tetanus toxoid.
Antitoxin antibodies work by neutralizing free toxin. However, toxin that is already bound to nerve endings cannot be neutralized with antibody.

Tetanus is preventable by immunization with tetanus toxoid. There is only one antigenic type of toxin, and immunization is safe and effective.

Question 13

What is the gram staining, sterilization method, and how many forms of C. botulinum are there?

Answer 13

Anaerobic, Gram-positive, spore-forming rod. Pressure sterilization is required to kill the spores. Seven antigenically distinct forms of botulism toxin (A-G) are described; A, B, E and F are associated with human disease. The toxins are phage-encoded (e.g., lysogenic conversion); each strain produces a single toxin type.

Question 14

What is the pathogenesis of C. botulinum poisoning?

Answer 14

Clinical manifestations of botulism are attributable to the action of botulinum toxin. This neurotoxin is one of the most potent toxins known. It is released during growth or sporulation of C. botulinum. Although C. botulinum spores remain viable for several hours at 100°C, preformed botulinum toxin is rather heat labile and is destroyed by boiling for 10 minutes or heating to 80°C for 30 minutes.
Botulism is similar in structure and function to tetanus toxin, except that botulinum is very specific for cholinergic nerves. The toxin blocks release of the neurotransmitter acetylcholine at the neuromuscular junction.

Question 15

What are the three forms of botulism?

Answer 15

(1) classic or food borne botulism
(2) infant botulism
(3) wound botulism.

Question 16

What is the clinical presentation of botulism?

Answer 16

Typical symptoms begin with blurred vision with fixed dilated pupils, dry mouth, constipation and abdominal pain. Flaccid paralysis develops with bilateral descending weakness of the peripheral muscles; death is attributed to respiratory paralysis.

Question 17

How is infant botulism acquired?

Answer 17

The most prevalent form of botulism in the US is associated with consumption of certain foods (esp. raw honey) by infants. Ingested spores germinate and produce toxin in the GI tract.

Question 18

How is botulism treated and prevented?

Answer 18

Botulism can be prevented by proper canning procedures to destroy contaminating spores, preventing spore germination in canned food by maintaining low pH, destroying preformed toxin by adequate heating.

Treatment includes
i) support ventilation, and
ii) polyvalent antitoxin.
Antitoxin neutralizes circulating toxin, but cannot reverse effects of toxin already bound to neurons. Recovery requires regeneration of affected nerve endings, and may take weeks to months.

Question 19

How would botulinum toxin most likely be used as a weapon?

Answer 19

The likely use of botulinum toxin as a bioweapon would be in an aerosolized or foodborne form.
Disease from aerosolized botulinum toxin is “inhalational botulism” and would result from absorption of inhaled, aerosolized toxin across the mucosal surface of the lung. Symptoms of disease are those of classical botulism. This mode of transmission has been:
• demonstrated experimentally in primates and as the result of laboratory accidents
• attempted by bioterrorists as recently as the three Japanese cult Aum Shinriky attacks in Japan between 1990 and 1995, and
• the emphasis of Iraq’s bioweapon development, including production of specially designed missiles and artillery shells for delivery.

Question 20

Describe the microbiology of C. perfringens

Answer 20

Gram-positive spore-forming rods, although spores are seldom seen in clinical specimens.
Gram-stain of wound exudates are notable for the absence of PMN’s due to the cytotoxic effects of alpha toxin.
C. perfringens has an exceptionally fast growth rate, with a generation time of 8 minutes.
The organism is relatively aerotolerant.

Question 21

How many toxins does C perfringens produce, what is the most important, and what does it do?

Answer 21

C. perfringens is known to produce 12 toxin that are active on tissues, plus an enterotoxin. The most important is alpha toxin, a phospholipase C (lecithinase) that cleaves phosphatidyl choline, a major lipid component of eukaryotic cell membranes. It is lyses a variety of cell types, including RBC’s, platelets, leukocytes and endothelial cells; resulting in destruction of vital tissue and widespread capillary damage.

Question 22

What diseases does C. perfringens cause?

Answer 22

1. Soft tissue infections include:
   a) cellulitis,
   b) fasciitis and
   c) myonecrosis (gas gangrene) in order of increasing damage to the surrounding tissue.
2. Clostridial food poisoning

Question 23

What is gas gangrene and how does it present?

Answer 23

Gas gangrene (clostridial myonecrosis) is a life-threatening, progressively destructive infection of a contaminated wound.
Sudden onset of pain around the wound, followed rapidly by discoloration, swelling, and the appearance of foul-sweet smelling watery exudate.
The usual setting is that of a crushing-type traumatic wound (gunshot, industrial and automobile accidents) in which there is considerable tissue damage and impaired bloodflow to the area (i.e. creation of an anaerobic environment).
Nearly half of incidents occur following bowel surgery.
Mortality is 40-60%.

Question 24

How is C. perfringens treated?

Answer 24

Treatment requires surgical debridement to remove dead tissue and antibiotic therapy. Hyperbaric oxygen may be useful since high oxygen tension inhibits growth and toxin production.

Question 25

How is C. difficile infection caused?

Answer 25

Antibiotic treatment
Alteration of the normal enteric flora allows either
1) overgrowth by endogenous C. difficile, or
2) makes the patient more susceptible to colonization and infection by exogenous organisms.
Historically, disease was thought to be uniquely associated with clindamycin usage, but virtually all antibiotics have now been implicated.

Question 26

How does C. difficile spread?

Answer 26

This is perhaps the only clostridial disease in which there is significant risk of person-to-person spread. Clustering of diarrhea and colitis cases in hospital wards has been shown to be associated with nosocomial spread of the organism to at-risk patients. Spread of the organism is facilitated by the ability of C. difficile to readily sporulate, and spores are resistant to alcohol-based hand sanitizer.

Question 27

What toxins does C difficile produce?

Answer 27

Disease is attributed to production of two toxins by C. difficile:
Toxin A (an enterotoxin) and
Toxin B (a cytotoxin).
Toxins are phage encoded, and only certain strains of C. difficile are lysogenized, produce toxin and capable of causing disease.

Question 28

How is C. difficile treated?

Answer 28

Treatment includes antibiotic therapy with an antimicrobial specifically effective against C. difficile, e.g., metronidazole (first line drug) and oral vancomycin (second-line drug). Prolonged treatment is indicated and recurrence of disease is not uncommon

Intestinal Microbiota Transplantation (Fecal Transplant). Used with patients with recurring C. diff disease in spite of prolonged antibiotic treatment is IMT.