Anaerobic Bacteria

Question 1

Common sources of anaerobic pathogen-normal flora

Answer 1

Normal flora:

• escape from normal compartment (Actinomyces, B corrodens, P. melaninogenica: mouse; B fragilis: colon and vagina
• establish abscesses
• abscess fills with bacteria and dead neutrophils, swells and becomes painful

Question 2

Common source of Pathogen- soil (spores)

Answer 2

-route of entry A- spores enter through wounds, germinate, and produce exotoxins- C. tetani, C. perfringens in gas gangre, C. botulinum- in wound botulism

Question 3

Common source of pathogen- soil (in food)

Answer 3

• route of entry B- spores germinate in vacuum packed foods
• C. perfringens in food poisoning: bacteria briefly survive in gut and release enteroxtoxin
• C. botulism infant botulism: bacteria briefly survive in gut and release neurotoxin
• C. botulinum in foodborne botulism- bacteria do not survive the gut, but have already filled food with neurotoxin

Question 4

Handling anaerobes in the laboratory

Answer 4

• sample must be handled anaerobically and labeled as such

- standard clinical labs are anaerobic culture, gram stain, gas chromatography

Question 5

Anaerobic Liquid Culture

Answer 5

• innoculate test tube of media
• add a reducing agent like thioglycolate to eliminate dissolved oxygen
• fill completely, stopper tightly and incubate without shaking
• strict aerobes on top- pseudomonas
• strict anaerobe on bottom- clostridum
• facultative anaerobe- E. coli

Question 6

Anaerobic culture streaking plates

Answer 6

• quickly streak onto agar media with usual benchtop sterile technique
• place plates in anaerobic culture jar
• add a chemical system to remove oxygen from the jar’s atmosphere and color indicate successful removed (Gas Pak)
• Airtight seal
• place whole jar in incubator for 48h
• note that colonies will die very shortly after removal from jar unless using a glovebox

Question 7

Identifying Anaerobes

Answer 7

• gram stain
• chemical testing (ability to ferment, hydrolize various macromolecules
• gas chromatography

Question 8

Anaerobic Infections: treatments

Answer 8

1) if abscess- surgical care: drain, debride
2) If toxigenic: antitoxin
3) Antibiotics: penicillin G, cefoxitin, chloramphenicol, clindamycin, metronidazole: specific for anaerobic bacteria, some protozoans

Question 9

Some anaerobic bacteria

Answer 9

• Clostridium- gram +, rod, spores
• Bacteroides- gram -, rod, no spores
• Prevotella- gram -, rod, no spores
• Actinomyces- gram +, long filamentous rod, no spores

Question 10

Some anerobic bacteria and their reservoirs and primary virulence factors

Answer 10

C. tetani- soil, tetanus, tetanospasmin
C. botulinum- soil, botulism, botulinum toxin
C. perfringens- soil, Gas Gangrene, Many tissue degrading enzymes
C. perfringens- soil, food poisoning, enterotoxin
C. difficile- normal flora, pseudomembranous colitis, exotoxins A and B
GNAB- Normal flora, Abscesses, a few tissue-degrading enzymes, capsule
Actiomyces spp- normal flora, Actinomycosis (abscess)

Question 11

Clostrudium tetani

Answer 11

-spores are environmental: soil, dust, manure, some human skin and GI
-gram +
-spore-forming
-transmitted to humans by soil contamination of wounds: splinters, thorns, punctures, IV drugs, septic surgery, septic handling of umbilical cord
-insertion beneath skin surface limits air contact
-spores germinate
-vegetative cells release exotoxin tetanospasmin
-

Question 12

Tetanospasmin

Answer 12

• 50kDa small subunit travels 2-14 days to spinal cord
• acts as protease, cleaves synaptobrevin in inhibitory motor nerves of the CNS
• without synaptobrevin, vesicles containing GABA and glycine cannot be released from affected neuron
• loss of central inhibitory activity on motor and autonomic neurons

Question 13

Generalized tetanus

Answer 13

• bacteria form a locus of infection
• exotoxin tetanospasmin enters bloodstream
• full-body symptoms cause morbidity
• > 50% untreated mortality from respiratory failure
• 21-31% treated mortality

Question 14

C. tetani prevention

Answer 14

-universal vaccination with tetanus toxoid( formaldehyde- treated toxin) in childhood (DTaP) according to standard schedule, adults get booster every 10 years

Question 15

C. difficile

Answer 15

• gram + spore forming rod
• causes pseudomembranous colitis= C. difficile- associated diarrhea (CDAD)= antibiotic-associated colitis= C. difficile colitis
• transmission:
• normal gut flora for 3% of general pop, 30% of hospitalized
• fecal oral- especially nosocomial from spores on hospital instruments or on hands of health care workers

Question 16

Pathogenesis of C. difficule

Answer 16

• recent course of antibiotics or cancer chemotherapeutics suppresses other normal flora, allows C. difficile to overgrow
• germinating cells release Exotoxin A, which disrupts tight junctions, causing intestinal swelling and inflammation
• exotoxin B is the major toxin, disrupts the cytoskeleton by depolymerizing actin, kills the surrounding cells
• a more virulent and drug-resistant strain of C. difficile emerged in 2001-2002; cases doubled 2000-2003 and have continued to rise since
• mortalitiy has increased from 6%

Question 17

C. difficile: diagnosis and treatment

Answer 17

• on sigmoidoscopy, patches of dead and dying cells appear as yellow-white plaques (pseudomembranes)
• withdraw initial antibiotic- cures 20%
• oral metronidazole or vancomycin
• surgical resection or removal of the colon may be required
• toxic megacolon or colonic perforation may occur