1. Bacteriodaceae, Clostridium, and the anaerobic cocci
The Anaerobes
Bacteriodaceae, Clostridium, and the anaerobic cocci

2. Bacteriodaceae Classification
Bacteroidaceae family includes the following genera
Bacteroides
Fusobacterium
Leptotrichia (rare in human diseases)
Prevotella
Porphyromomas
B. fragilis is the most commonly isolated anaerobic G-B.
All are nonsporing, anaerobic, G-B

3. Bacteriodaceae Morphology/cultural characteristics
They may be NF of the oropharynx, urogenital tract, and colon and are considered opportunistic pathogens
Morphology/cultural characteristics
Pleomorphic G-B showing irregular or bipolar staining.
Fusobacterium nucleatum characteristically is long and slender with pointed ends

4. Bacteroides sp. Gram stain

5. Fusobacterium sp.

6. Fusobacterium nucleatum

7. Bacteriodaceae
To grow these organisms, nonselective anaerobic BA plates, selective anaerobic plates, and liquid should be used for primary isolation.
Nonselective anaerobic BA plates= CBA plates plus vitamin K1, hemin, yeast extract, and L-cystine (supplemented BA)
Selective media (all for Bacteroides species)
Anaerobic PEA BA – suppresses aerobic G-B
Kanamycin-Vancomycin BA – inhibits G+ and facultatively anaerobic G -

8. Bacteriodaceae Must incubate under strict anaerobic conditions
Kanamycin-Vancomycin laked BA (KVLB) prepared by freezing and thawing whole blood
Bacteroides Bile Esculin (BBE) agar
Liquid media
Thioglycollate
Chopped meat glucose
Must incubate under strict anaerobic conditions
Incubation at C for 48 hours before opening an anaerobic jar.

9. B. fragilis

10. Bacteriodaceae Biochemistry
Each colony type that grows should be Gram stained and subcultured to plates grown under both aerobic and anaerobic conditions to confirm that it is an anaerobe.
Biochemistry
The Bacteroides group which now includes Prevotella and Porphyromonas species are divided into groups based on bile tolerance, pigment production, and sensitivity to the antibiotics Vancomycin (V), Kanamycin (K), and Colistin (C)

11. Bacteriodaceae V K C Bile pigment Bacteroides fragilis R R R R -
Prevotella R R S S /-
Porphyromonas S R R S
B. fragilis is catalase +
GLC – used to differentiate Fusobacterium from the others.
The major by-product of Fusobacterium is butyric acid while the others produce mixtures of acids.
F. nucleatum and F. necrophorum (lipase+) are the major pathogens

12. GLC

13. Bacteriodaceae Virulence factors Clinical significance
Fusobacterium – endotoxin; the endotoxin of Bacteroides is not highly toxic
B. fragilis – capsule
Some in the Bacteroides group produce IgA protease,collagenase, phosphotase, RNAse, or DNAse
Clinical significance

14. Bacteriodaceae Clinical significance
These organisms are NF of the oropharynx, urogenital tract, and colon and cause serious infections when they gain access to tissues and organs.
Most commonly they cause intra-abdominal infections
The infections are usually polymicrobial infections
They can also be found causing:
Peridontal disease

15. Bacteriodaceae Antimicrobial susceptability/treatment
Chronic otitis media
Chronic sinusitis
Wound infections
Pneumonia
Female genital tract infections
Brain abscesses
Bacterial endocarditis
Bone infections
Antimicrobial susceptability/treatment
Incision and drainage
Chloramphenicol, clindamycin, cefoxitin, or metronidazole

16. Clostridium Classification – no family designation
Most are strict anaerobes
Are widely distributed in soil and water
Some are NF in the GI tract of man and other animals
Morphology/cultural characteristics
Are endospore forming large G+B
The site at which the endospore forms in the vegetative cell is characteristic and helps in differentiating C. tetani (terminal) from the others (oval and subterminal)

17. C. perfringens Gram stain

18. Spore stain with time

19. C. tetani spores (terminal)

20. Clostridium All except C. perfringens are motile
Nonselective, selective, and liquid media should be used for primary isolation
Nonselective – supplemented anaerobic BA
C. perfringens produces a classic double zone of hemolysis
Nonselective, differential – Egg yolk agar
Allows differentiation based on
Lecithinase production (white precipitate)
Lipase production (sheen around surface of colonies)
Protease production(clearing)

21. C. perfringens double zone hemolysis

22. Lecithinase production

23. Lipase production

24. Clostridium
Selective – Cycloserine-cefoxitin-egg yolk-fructose agar(CCFA) is selective for C. difficile
Liquid
Thioglycollate
Chopped meat
Special isolation procedures – Clostridia usually occur in mixed cultures with G-B and nonsporing anaerobes – use heat or alcohol treatment to kill others before plating
C. perfringens grows rapidly at 450 C

25. CCFA

26. Clostridium Biochemistry
O2 tolerance – C. tertium and C. histolyticum are aerotolerant, but catalase -
Lipase vs lecithinase vs protease production on egg yolk agar
Naegler reaction - smear ½ of an egg yolk agar plate with type A anti-toxin (anti-lecithinase), streak organism in a single line, and look for inhibition of lecithinase production
Sugar fermentations

27. Clostridium
Milk digestion
Esculin hydrolysis
Gelatin hydrolysis
Reverse CAMP – is presumptive for C. perfringens
Mechanisms of Virulence – most Clostridia are not invasive, but many produce powerful toxins and enzymes
C. perfringens produces a capsule
C. botulinum – produces a potent exotoxin
The organisms are divided into 8 different types based on which of the 8 serotypes of exotoxin are produced.

28. Clostridium
Serotypes are A, B, C1, C2, D, E, F, and G
Serotype A is the most potent.
Types A,B, E, F, and G can cause botulism in man
Botulinal exotoxin is the most powerful exotoxin known.
It works at the neuromuscular junction and in the autonomic nervous system to prevent the release of the neurotransmitter acetylcholine.
This leads to flaccid paralysis.
The toxin has two components, B for binding, and A for the toxic activity.
The toxic part cleaves proteins that mediate fusion of synaptic vesicles with the cell membrane and subsequent release of acetylcholine.
The toxin is part of the bacteria and not released until the death of the bacteria.

29. Normal release of acetylcholine

30. Proteins involved in membrane fusion to release acetylcholine

31. C. botulinum toxin prevents acetylcholine release

32. Botox treatment – relaxes muscle spasms

33. Clostridium C. tetani -produces two exotoxins A hemolysin
Tetanospasmin – can travel to the CNS humorally through blood and lymph, or neurally through tissue spaces of the peripheral nerves.
The toxin binds to sialic acid containing gangliosides of the CNS to prevent the release of the inhibitory neurotransmitters GABA and glycine from synapses (by cleaving VAMP) in the inhibitory nerve system of the spinal cord.
This is a system that prevents the contraction of a muscle when the muscle of the opposite action contracts.
This leads to both sets of muscles contracting at the same time and spastic paralysis.
Called lockjaw when the jaw is affected.
Contractions can break the back.

34. Tetanospasmin activity

35. Spastic paralysis

36. Tetanospasmin and botulinism toxin action

37. Clostridium
C. perfringens – is divided into 5 types, A through E, based on the major lethal toxins produced.
All types produce the alpha toxin – is a lethal, necrotizing lecithinase which is responsible for the outer zone of hemolysis.
Other toxins that may be produced include beta, epsilon, or iota which are all lethal and necrotizing, delta and theta which are lethal and hemolytic and are responsible for the inner zone of hemolysis.

38. Clostridium
Enzymes produces may include gelatinase, collagenase, protease, hyaluronidase, DNAse, and neuraminidase
Types A and C produce an enterotoxin responsible for causing an intoxication type of food poisoning in meats, poultry, and gravy.
Its action resembles that of the cholera toxin.

39. Clostridium
C. difficile – produces two exotoxins both of which inactivate Rho proteins by adding a glucose.
Rho proteins function as molecular switches in cytoskeletal dynamics and many signal transduction pathways
Enterotoxin A stimulates fluid and electrolyte losses from the intestinal tract
Cytotoxin B kills mammalian cells

40. Clostridium Clinical significance
C. tetani – causes tetanus, a disease due to the toxin tetanospasmin.
Spores are found in feces, soil, and dust. Spores enter the body through wounds where they germinate into vegetative cells and subsequently produce toxin when a sufficiently low O/R potential is established in the infected tissue (usually a deep wound).

41. Clostridium
The incubation is 1-54 days with an average of 6-15 days. The longer the incubation, the better the prognosis.
Symptoms begin with cramps and twitching of muscles around the wound.
Headache and neck stiffness also occur.
These are followed by trismus (lockjaw) and more generalized symptoms.
Death, if it occurs, results from respiratory failure within 4 days.
Neonatal tetanus is a consequence of infection of the umbilicus through septic midwifery and it occurs in underdeveloped countries.

42. Tetanus

43. Clostridium C. botulinum – causes botulism
Food botulism – in the U.S. this usually occurs following ingestion of inadequately processed home-canned food. For this to occur you need:
Food must be contaminated with C. botulinum spores
Food must possess composition and nutritive properties that allow germination and toxin production
Food must have suitable pH and temperature
Food must have been inadequately heated or processed (toxin is heat labile)

44. Clostridium
Following ingestion, toxin is absorbed from the intestine and transported via blood and lymph to the PNS.
The incubation is 8 hours to 8 days with hours most common
The first symptoms include nausea, vomiting, and diarrhea followed by symmetric, descending paralysis (eyes, throat, neck, trunk, and then the limbs)
Paralysis of respiratory muscles results in death
Infant botulism – follows ingestion of spores which germinate in the intestine.

45. Clostridium
Illness may range from subclinical to sudden infant death syndrome
Honey has been implicated as a source of spores.
This doesn’t occur in adults because of competing NF
Wound botulism – can follow C. botulinum toxin production in a traumatic wound.
Clostridial wound infections – most species of Clostridium are saprophytic bacteria living in soil and water.
C. perfringens and other are found in the intestines of man and other animals.

46. Clostridium
Most clostridial wound infections occur as simple contaminants of a fresh wound and most heal normally with simple therapy
Anaerobic cellulitis follows invasion of necrotic wound tissue by proteolytic bacteria and is characterized by gas accumulation, discoloration of the skin, and a malodorous brown, purulent discharge.
Clostridial myonecrosis or gas gangrene – involves invasion of normal, healthy muscle tissue surrounding the wound site.
C. perfringens is most commonly isolated. Need a lowered O/R potential in the wound which causes reduction of pyruvate to lactate and a decreased pH which activates the proteolytic enzymes.
Clinical features include severe systemic toxicity, a painful, edematous wound with a sweet or foul smelling discharge.
Untreated cases may result in death.

47. Gas gangrene

48. Clostridium
C. difficile – Antibiotic therapy of any kind can result in diarrhea.
The severity may range from simple diarrhea to severe antibiotic associated pseudomembranous colitis.
This is characterized by colonic plaques that coalesce to form a pseudomembrane of mucin, fibrin, sloughed off epithelial cells, and acute inflammatory cells.
Complications include dehydration, electrolyte imbalance and colonic perforation.
Most often occurs following therapy with ampicillin, clindamycin, or cephalosporins and is usually due to C. difficile which may be NF in the G.I. tract.

49. Pseudomembranous colitis plaques

50. Pseudomembranous colitis plaques

51. Clostridium Antimicrobial therapy/treatment
C. tetani - use antitoxin, however, toxin already in the CNS is not neutralized; maintain open airway; remove infected tissue.vaccination - part of DPT – need booster every 10 years
C. botulinum – administer antitoxin before it is translocated inside tissues: aggressive respiratory support
Wound infections – debridement and antibiotics; hyperbaric chamber

52. Clostridium
C. difficile – metronidazole and vancomycin; fluid and electrolyte replacement

53. Anaerobic cocci Classification Morphology and general characteristics
Peptococcus
Peptostreptococcus – anaerobic coccus most often associated with human disease
Veillonella
All can be found as NF of oral cavity, Genital and urinary tracts, G.I. tract , and skin
Morphology and general characteristics
Peptococcus – G+C; 1 species, P. niger, produces black colonies
Peptostreptococcus G+C; 9 species. P. anaerobius is the most commonly isolated species

54. Peptostreptococcus Gram stain

55. Anaerobic cocci Biochemisrty
Veillonella – tiny G-C; V. parvula is the major isolate of clinical importance.
All are slow growing and may require prolonged incubation
Biochemisrty
I.D. by GLC
P. anaerobius is presumptively identified based on sensitivity to polyanethol sulfonate (SPS)

56. Anaerobic cocci Clinical significance
Usually in polymicrobial infections with aerobic organisms and
Caused from spread from a site they normally colonize to an adjacent sterile site
Cause pleuropulmonary infections, sinusitis, brain abscesses, intra-abdominal infections, pelvic infections, endocarditis, and osteomyelitis

57. Anaerobic cocci Antimicrobial therapy
Cephalosporins, clindamycin, and chloramphenicol