culture

1. Anae rob ic Bacte ria

2. Caegor
t y
 Spore-forming:
rod, Gram (+)---
Clostridium
 Nonspore-forming:
see next
slides

3. Caegor
t y
Spore- rod, Gram (+)---
forming: Clostridium
Nonspore-forming:
Rod, Gram (+) Propionibacterium 丙酸菌属
Bifidobacterium
Lactobacillus
Eubacterium
Rod, Gram (-) Bacteroides Actinomyces
Fusobacterium 梭菌属
Cocci, Gram (+) Peptococcus
Campylobacter
Peptostreptococcus
Cocci, Gram (-) Veillonella

4. Clostridium Species
 The clostridia are opportunistic
pathogens. Nonetheless, they are
responsible for some of the deadliest
diseases including gas gangrene,
tetanus and botulism. Less life-
threatening diseases include
pseudomembranous colitis (PC) and
food poisoning.
 cause disease primarily through the
production of numerous exotoxins.
 perfringens, tetani, botulinum, difficile

5. Clostridium Tetani
Pathogenesis of tetanus caused by C tetani

6. General introduction
 C tetani is found worldwide.
Ubiquitous in soil, it is occasionally
found in intestinal flora of humans
and animals
 C.tetani is the cause of tetanus,or
lockjaw. When spores are introduced
into wounds by contaminated soil or
foreign objects such as nails or glass
splinters

7. BIOCHEMICAL CHARACTERISTICS
 Morphology: long and slender;
peritrichous flagella,no capsule,
terminal located round
spore(drum-stick apperance),
its diameter greater than
vegetative cell.
 Culture:obligate anaerobic;
Gram(+); swarming occures on
blood agar, faint hemolysis.
 Biochemical activities:does not
ferment any carbohydrate and
proteins.
 Resistance: tolerate boiling for
60 min.alive several ten years in 2-5 x 0.3-0.5um
soil.
 Classification and Antigenic
Types: C tetani is the only
species. There are no serotypes

8. Pathogenicity
 No invasiveness; toxemia  retrograde transport
(exogenous infection ） to (CNS)
 produces two exotoxins:  delitescence ： a few
tetanolysin, and
tetanospasmin(a kind of days to several
neurotoxin, toxicity weeks
strong)  The two animal
 The actions of species most
tetanospasmin are
susceptible to this
complex and involve three
components of the toxemia are horses
nervous system: central and humans.
motor control, autonomic
function, and the
neuromuscular junction.

9. Clostridium tetani -Tetanospasmin
 disseminates systemically
 binds to ganglioside receptors
• inhibitory neurones in CNS
 glycine
• neurotransmitter
 stops nerve impulse to muscles
 spastic paralysis 痉挛性麻痹
 severe muscle contractions and
spasms
 can be fatal

10. Tetanospasmin

11. Clinical Manifestations
 The initial symptom is cramping and
twitching of muscles around a wound. The
patient usually has no fever but sweats
profusely and begins to experience pain,
especially in the area of the wound and
around the neck and jaw muscles (trismus).
 Portions of the body may become extremely
rigid, and opisthotonos 角弓反张 (a spasm
in which the head and heels are bent
backward and the body bowed forward) is
common.
 Complications include fractures, bowel
impaction, intramuscular hematoma, muscle
ruptures, and pulmonary, renal, and cardiac
problems

12. Clinical Manifestations
DISEASE CLINCAL MANIFESTATIONSA
Generalized Involvement of bulbar and paraspinal
muscles(trismus or lockjaw, risus sardonicus,
difficulty swallowing, irritability,
opisthotonos);involvement of autonomic
nervous system(sweating, hyper thermia,
cardiac arrhythmias, fluctuations in blood
pressure)
Cephalic Primary infection in head,particularly
ear;isolated or combined involvement of cranial
nerves, particularly seventh cranial nerve; very
poor prognosis
Localized Involvement of muscles in area of primary
injury; infection may precede generalized
disease; favorable prognosis
Neonatal Generalized disease in neonates; infection
typically originates from umbilical 脐带
stump;very poor prognosis in infants whose
mothers are nonimmune

14. Epidemiology
 1 million cases of tetanus occur annually in the
world,with a mortality rate ranging from20%
to 50%. But rare in most developed countries.
 In some developing countries, tetanus is still
one of the ten leading causes of death, and
neonatal tetanus accounts for approximately
one-half of the cases worldwide.
 In less developed countries, approximate
mortality rates remain 85% for neonatal
tetanus and 50% for nonneonatal tetanus.
 In the United States, intravenous drug abusers
have become another population with an
increasing incidence of clinical tetanus
 In untreated tetanus, the fatality rate is 90%
for the newborn and 40% for adults.

15. Immunity
 Humoral immunity(antitoxin)
 There is little, if any, inate immunity
and the disease does not produce
immunity in the patient.
 Active immunity follows vaccination
with tetanus toxoid

16. Diagnosis
 Diagnosis is primarily by the clinical
symptoms (above). The wound may not
be obvious.
 C tetani can be recovered from the
wound in only about one-third of the
cases.
 It is important for the clinician to be
aware that toxigenic strains of C tetani
can grow actively in the wound of an
immunized person.
 Numerous syndromes, including rabies
and meningitis, have symptoms similar
to those of tetanus and must be
considered in the differential diagnosis.

17. Vaccination
• infant
• DPT (diptheria, pertussis, tetanus)
• tetanus toxoid
– antigenic
– no exotoxic activity

18. Control
 The offending organism must be
removed by local debridemen 清创
术
 toxoid
 TAT; Metronidazole (For more
serious wounds)
 AIDS patients may not respond
to prophylactic injections of
tetanus toxoid

19. C. perfringens
• soil, fecal contamination
• gas gangrene
– swelling of tissues
– gas release
* fermentation products
• wound contamination

20. Toxins
toxin Biological Feature Types of Toxins
A B C D E
α lecithinase; increase the
vascular permeability; + + + + +
hemolytic; produces
necrotizing activity
β Necrotizing activity, － + + － －
induces hypertension
by causing release of
catecholamines.
ε increase the － － － + －
permeability of
gastrointestinal wall
τ Necrotizing activity;
increase the vascular
－ － － － +
permeability

21. Toxins
 Many of these toxins have lethal,
necrotizing, and hemolytic properties;
 The alpha toxin produced by all types of C.
perfringens, is a lecithinase that lyses
erythrocytes, platelets, leukocytes, and
endothelial cells. And its lethal action is
proportionate to the rate at which it splits
lecithin to phosphorylcholine and
diglyceride.
 The theta toxin has similar hemolytic and
necrotizing effects.
 DNAase, hyaluronidase, a collagenase are
also produced

22. Enterotoxin
 Many strains of type A produce
enterotoxin, which is a heat-labile protein
and destroyed immediately at 100 ℃.
 Trypsin treatment enhances the toxin
activity threefold.
 The toxin is produced primarily by type A
strains but also by a few type C and D
strains.
 It disrupts ion transport in the
ileum(primarily) and jejunum by inserting
into the cell membrane and altering
membrane permeability.
 As superantigen.

24. Pathogenesis
•Tissue degrading enzymes
– lecithinase [l toxin]
– proteolytic enzymes
– saccharolytic enzymes
• Destruction of blood vessels
• Tissue necrosis
• Anaerobic environment created
• Organism spreads

25. Without treatment death
occurs within 2 days
 effective antibiotic therapy
 debridement
 anti-toxin
 amputation & death is rare

26. Gas gangrene
 Gas gangrene is a life-threatening disease with
a poor prognosis and often fatal outcome.
 Initial trauma to host tissue damages muscle and
impairs blood supply----lack of oxygenation
 Initial symptoms : fever and pain in the infected
tissue.; more local tissue necrosis and systemic
toxemia. Infected muscle is discolored (purple
mottling) and edematous and produces a foul-
smelling exudate; gas bubbles form from the
products of anaerobic fermentation.

27. Gas gangrene
 As capillary permeability increases,
the accumulation of fluid increases,
and venous return eventually is
curtailed.
 As more tissue becomes involved,
the clostridia multiply within the
increasing area of dead tissue,
releasing more toxins into the local
tissue and the systemic circulation.

28. Food poisoning
 Enterotoxin producing strains.
 These bacteria are found in
mammalian faeces and soil.
 Small numbers of the bacteria may
also be found in foods and they may
propagate rapidly to dangerous
concentrations if the food is
improperly stored and handled.

29. Food poisoning
 more than 108 vegetative cells are
ingested and sporulate in the gut, the
toxins can act rapidly in the body,
causing severe diarrhea in 6-18 hours,
dysentery, gangrene, muscle infections
 The action of C. perfringens enterotoxin
involves marked hypersecretion in the
jejunum and ileum, with loss of fluids
and electrolytes in diarrhea.

30. Cellulitis, Fasciitis
 Cellulitis, Fasciitis
 Fasciitis : a rapidly progressive,
destructive process in which the
organisms spread through fascial plan es.
 Fasciitis causes suppuration and the
formation of gas
 Absense of muscle involvement
 rapidity

31.  Necrotizing Enteritis
 Rare, acute necrotizing process in the
jejunum
 Abdominal pain, bloody diarrhea, shock, and
peritonitis
 Mortality: 50%
 Beta-toxin-producing C. perfringens type C
 Septicemia

32. Who is at risk?
 Surgical patients; patient after
trauma with soil contamination.
 People who ingest contaminated
meat products (without proper
refrigeration or reheating to
inactivate endotoxin)

33. Epidemiology
 C. perfringens type A: the intestinal
tract of humans and animals, soil
and water contaminated with feces.
forms spores under adverse
environmental conditions and can
survive for prolonged periods.
 Type B to E strains colonize the
intestinal tract of animals and
occasionally humans.

34. Epidemiology
 Type A: gas gangrene, soft tissue
infections and food poisoning
 Type C: enteritis; necroticans

35. Laboratory identification
• lecithinase production
Double Hemolysis Circles

36. C. botulinum

37. Biological Features
 Anaerobic
 Gram-positive
 rod-shaped
 sporeformer
 produces a protein neurotoxic.
 soil, sediments of lakes, ponds,
decaying vegetation.
 intestinal tracts of birds, mammals
and fish.

38. Division
---A, B, C1, D, E, F, and G.
---type A. 62%
---Not all produce toxin.
---C and D not
---G plasmid encoded.

39. Transmission
---spores heat resistant.
canning.
anaerobic environment
---Botulism
eating uncooked foods
spores
---GI, duodenum, blood stream,
neuromuscular synapses.

40. Virulence factors
---bacterial protease
---light chain,A,50 kDa;
heavy chain,100kDa.
---disulfide bond.
---A potent toxin

41.  binds peripheral nerve receptors
• acetylcholine neurotransmitter
 inhibits nerve impulses
 flaccid paralysis
 death
• respiratory Botulinum toxin
• cardiac failure

42. Botulinum toxin
 Bioterrorism
• not an infection
• resembles a chemical attack
• 10 ng can kill a normal adult

43. Epidemiology
---4: foodborne, infant, wound, undetermined.
---Certain foods; wound not.
---Foodborne botulism, consumption.
---Infant botulism, 1976, under 12m.
---ingestion, colonize and produce toxin in the
intestinal tract of infants.
honey.
---increased.
---internationally recognized.

44. Clinical syndromes
---18-36 hours:
---weakness, dizziness,dryness of the mouth.
---Nausea,vomiting.
---Neurologic features: blurred vision,
inability to swallow, difficulty in speech,
descending weakness of skeletal muscles,
respiratory paralysis.

45. Botulism( 肉毒中毒 )
 food poisoning
• rare
• fatal
 germination of spore
 inadequately sterilized canned food
• home
 not an infection

46. Infection with C. botulinum
 Neonatal botulism
• uncommon
• the predominant form of
botulism
• colonization occurs
 no normal flora to compete
 unlike adult

47. Wounds
• extremely rare
• an infection

48. Immunity
---specifically neutralized, antitoxin.
---toxoided, make good antigens.
---does not develop, amount toxic.
---Repeated occurrence.
---Once bound, unaffected by antitoxin.
---circulating toxin ,neutralized , injection
of antitoxin.
---treated immediately with antiserum.
---multivalent
toxoid,unjustified,infrequency.
experimental vaccine.

49. Diagnosis
---by clinical symptoms alone
---differentiation difficult.
--- most direct and effective: serum or
feces.
---most sensitive and widely used:
mouse neutralization test. 48h.
Culturing of specimens 5-7d.

50. Treatment
 Individuals known to have ingested food
with botulism should be treated
immediately with antiserum.
 antibiotic therapy (if infection)
• Vaccination will not protect hosts
from botulism, however passive
immunisation with antibody is the
treatment of choice for cases of
botulism.

51. Prevention
---proper food handling and preparation.
--- spores survive boiling (100 degrees
at 1 atm) 1h.
---toxin heat-labile, boiling or intense
heating, inactivate the toxin.
---bulge, gas, spoiled.

52. C. difficile
• After antibiotic use
• Intestinal normal flora --greatly decreased
• Colonization occurs
• Enterotoxin secreted
• Pseudomembanous colitis

53. Pseudomembranous Colitis
 Pseudomembranous colitis (PC) results
predominantly as a consequence of the
elimination of normal intestinal flora
through antibiotic therapy.
 Symptoms include abdominal pain with
a watery diarrhea and leukocytosis.
"Pseudomembranes" consisting of
fibrin, mucus and leukocytes can be
observed by colonoscopy.
 Untreated pseudomembranous colitis
can be fatal in about 27-44%.

54. Therapy
 Discontinuation of initial antibiotic
(e.g. ampicillin)
 Specific antibiotic therapy (e.g.
vancomycin)

55. Obligate (strict) anaerobes
• no oxidative phosphorylation
• fermentation
• killed by oxygen
• lack certain enzymes
– superoxide dismutase
* O2-+2H+ H2O2
– catalase
* H2O2 H20 + O2
– peroxidase
* H2O2 H20 /NAD to NADH

56. Strict anaerobe infectious
disease
 Sites throughout body
 Muscle, cutaneous/sub-cutaneous
necrosis
 Abscesses

58. Bacterial Flora of the Body
Site Total Bacteria Ratio
(per/ml or gm) Anaerobes:Aerobes
Upper Airway
Nasal Washings 103-104 3-5:1
Saliva 108-109 1:1
Tooth Surface 1010-1011 1:1
Gingival Crevice 1011-1012 1000:1
Gastrointestinal Tract
Stomach 102-105 1:1
Small Bowel 102-104 1:1
Ileum 104-107 1:1
Colon 1011-1012 1000:1
Female Genital Tract
Endocervix 108-109 3-5:1
Vagina 108-109 3-5:1

59. Problems in identification of
anaerobic infections
• air in sample (sampling, transportation)
– no growth
• identification takes several days or longer
– limiting usefulness
• often derived from normal flora
– sample contamination can confuse

60. Virulence Factors
1. Anti-phagocytic capsule
• Also promote abscess formation
2. Tissue destructive enzymes
• B. fragilis produces variety of enzymes
(lipases, proteases, collagenases) that
destroy tissue  Abscess Formation
3. Beta-lactamase production
• B. fragilis – protect themselves and other
species in mixed infections
4. Superoxide dismutase production
• Protects bacteria from toxic O2 radicals as
they move out of usual niche

61. Characteristics of Anaerobic Infections
1. Most pathogenic anaerobes are
usually commensals
• Originate from our own flora
2. Predisposing Conditions
• Breeches in the mucocutaneous barrier
  displace normal flora
• Compromised vascular supply
• Trauma with tissue destruction
• Antecedent infection

62. Characteristics of Anaerobic
Infections
3. Complex Flora 4. Synergistic Mixture
 Multiple species of Aerobes &
• Abdominal Infection  Avg Anaerobes
of 5 species
 3 anaerobic
 E. coli  Consume O2
 2 aerobic • Allow growth of
• Less complex then nl flora anaerobes
• Fecal flora 400 different
species  Anaerobes  promote
 Those predominant in stool growth of other
are not infecting species
bacteria by being
• Veillonella,
Bifidobacterium  rarely antiphagocytic and
pathogenic
producing B-
• Species uniquely suited to
cause infection lactamases
predominate

63. Clues to Anaerobic Infection
• Infections in continuity to mucosal
surfaces
• Infections with tissue necrosis and
abscess formation
• Putrid odor
• Gas in tissues
• Polymicrobial flora
• Failure to grow in the lab
BIOCHEMICAL KITS
• e.g. API SYSTEM
GAS CHROMATOGRAPHY
• volatile fermentation products

64. Bacteroides fragilis
• Major disease causing strict anaerobic
after abdominal surgery
non-spore-former
• Prominent capsule
– anti-phagocytic
– abscess formation
• Endotoxin
– low toxicity
– structure different than other
lipolysaccharide

65. • Enterobacteriaceae (facultative anaerobes)
– commonly cause disease
– low numbers gut flora
• Strict anaerobes
– much less commonly cause disease
– high numbers gut flora .