1. Anaerobes of clinical Importance

2. Oxygen
Class Definition Examples
Obligate aerobe Grow only in Mycobacterium
the presence of tuberculosis
O2
Obligate anaerobe Can not grow in Clostridia
the presence of
O2
Facultative Can grow in the Most bacteria of
anaerobe presence or medical
absence of O2 importance
Microaerophilic Require low O2 Campylobacter
bacteria tension

3. 2H 2O + 2O 2 = 2H 2O 2 + O −
2
In the presence of oxygen, two toxic substances to
the bacteria are produced which are hydrogen peroxide
and superoxide anion.
In obligate aerobes and facultative anaerobes:
Catalase and peroxidase enzymes degrade hydrogen
peroxide.
Superoxide dismutase enzyme degrades superoxide anion.
BUT
In obligate anaerobes:
These enzymes are not present.
So, the presence of oxygen is toxic to them.

4. CLASSIFICATION
Anaerobic spore forming bacilli (Clostridia)
Gram negative bacilli non-sporing forming (Bacteroides)
Anaerobic streptococci (Peptostreptococcus)
Anaerobic staphylococcus (Peptococcus)
Gram negative diplococci (Veillonella)
Gram positive bacilli (Actinomyces)

5. :HABITAT I
These organism are normal flora in:
• A. Oropharynx eg. . Fusobacteria and Veillonella
• B. Gastrointestinal tract
– Found mainly in the large colon in large numbers
– Total number of anaerobes = 10 11
– While all aerobes (including E. coli) = 10 4
– examples are (1) B acteroides fragilis (2) Bifidobacterium
species
• C. Female genital tract (mainly in the vagina)

6. Features of anaerobic infections
• Infections are always near to the site of the
body which are habitat.
1. Infection from animal bites.
2. Deep abscesses
3. The infections are also polymicrobial
4. Gas formation, foul smell
5. Detection of "Sulphur granules"' due to actinomycosis
6. Failure to grow organism from pus if not culture
anaerobically.
7. Failure to respond to usual antibiotics.

7. Infections begin :
• Disruption of barriers
– Trauma
– Operations
– Cancerous invasion of tissues
• Disruption of blood supply
– Drops oxygen content of tissue
– Tissue necrosis

8. what are the infection caused by
these anaerobic organisms
Post operative wound infection
Brain, dental, lung abscess
Intra abdominal abscess, appendicitis, diverculitis
 Infection of the female genital tract: Septic abortion,
endometritis , , puerperal infection and endometritis ,
pelvic abscess or breast abscess
Diabetic foot infections

9. :Laboratory diagnosis
• When anaerobic infection is suspected;
a) Specimens have to be collected from the site containing
necrotic tissue.
b) Pus is better than swabs.
c) Specimens has to be send to the laboratory within 1/2
hour
d) Fluid media like cooked meat broth are the best culture
media.
e) Specimens have to incubated anaerobically for 48 hours.

10. Organism groups
Gram negative rods
Bacteroides
Prevotella
Porphyromonas
Fusobacterium
Butyrivibrio
Succinomonas

11. Bacteroides
Strict anaerobe
Pleomorphic
Gram negative bacilli (cocco bacilli)
Normal flora in
Oropharynx
 Gastrointestinal tract
Vagina

12. Bacteroides
• B. Fragilis, B. Vulgaris, B. Uniformis
– Account for 1/3 of all isolates
– Resistant to 20% bile
– Resistant to many antibiotics
• Penicillin, kanamycin, vancomycin,
colistin – and many more
– No pigmentation of colonies or fluorescence
•

13. Bacteroides other species
Bacteroides species other than b. Fragilis
group
Bile sensitive
Resistant to kanamycin only
Some pigmented

14. Other gram negative rods
• Fusobacterium necrophorum
• Gram negative bacilli
• Peritonisillar intrnal jugular vein thrombosisemboli
to the lung
• Peptococcus
• Gram positive cocci in clusters
• Peptostreptococcus
• Gram positive cocci in chains
• Brain abscess
• Veillonella parvula
• Gram negative cocci

15. Clostridium SPP

16. Clostridium spp. Anaerobic Gram-Positive Spore-Forming Bacilli
Four broad types of pathogenesis:
1. Histotoxic group — tissue infections
(C. perfringens type A, exogenously acquired more commonly than endogenously)
(C. septicum; endogenously-acquired)
a. cellulilitis
b. myonecrosis
c. gas gangrene
d. fasciitis
2. Enterotoxigenic group — gastrointestinal disease
a. clostridial foodbome disease (8-24h after ingestion of large numbers of organisms on con-taminated
meat products, spores germinate, enterotoxin produced (C. perfringens type A)
b. necrotizing enteritis (beta toxin-producing C.perfringens type C)
(C. difficile endogenously-acquired or exogenously-acquired person-to-person in hospital)
c. antibiotic-associated diarrhea
d. antibiotic-associated pseudomembrane colitis
3. Tetanus (exogenously acquired) — C. tetani neurotoxin
a. generalized (most common)
b. cephalic(primary infection in head, comnnonly ear)
c. localized
e. neonatal (contaminated umbilical stump)
4. Botulism (exogenously acquired) — C. botulinum neurotoxin
a. foodborne (intoxication,1-2days incubation period)
b. infant (ingestion of spores in honey)
c. wound (symptoms similar to foodborne, but 4 or more days incubation)

17. Spores
 Clostridium form endospores under unfavourable
environmental conditions
 Spores are a survival mechanism
 Spores are characterized on the basis of position,
size and shape
 Most Clostridium spp., including C. perfringens and
C. botulinum, have ovoid subterminal near an end) (
(OST) spores
 C. tetani have round terminal (RT) spores

18. Clostridium Associated Human Disease

19. Clostridium perfringens

20. Clostridium perfringens — histotoxic or enterotoxigenic infections
Morphology and Physiology
• large, rectangular bacilli (rod) staining gram-positive
• spores rarely seen in vitro or in clinical specimens (ovoid, subterminal)
• non-motile, but rapid spreading growth on blood agar mimics growth of motile organisms
• aerotolerant, especially on media supplemented with blood
• grow at temperature of 20-50°C (optimum 45°C) and pH of 5.5-8.0
Pathogenicity Determinants (note that toxins include both cytolytic enzymes and bipartite exotoxins)
• four major lethal toxins (alpha (α), beta (β), epsilon (ε), and iota (ι) toxins) and an enterotoxin
• six minor toxins (delta(δ), theta(θ), kappa(κ), lambda(λ), mu(µ), nu(η)toxins) & neuraminadase
• C. perfringens subdivided into five types (A-E) on basis of production of major lethal toxins
• C. perfringens Type A (only major lethal toxin is alpha toxin) responsible for histotoxic and
enterotoxigenic infections in humans; Type C causes necrotizing enteritis (not in U.S.)
Lab Identification
• direct smear and Gram stain, capsules upon direct examination of wound smears
• culture takes advantage of rapid growth in chopped meat media at 45° C to enrich and then
isolate onto blood agar streak plate after four to six hours
• gas from glucose fermentation
• in vivo toxicity testing and identification of the specific toxin types involved
• double zone of hemolysis on blood agar (p-hemolytic theta(e) toxin, a-hemolytic alpha(oc) toxin)
• Nagler rxn; precipitation in serum or egg yolk media; oc -toxin (phospholipase C) is a lecithinase
• "stormy" fermentation (coagulaltion) of milk due to large amounts of acid and gas from lactose
Diagnosis/Treatment of systemic infection — Early diagnosis and aggressive treatment essential
• removal of necrotic tissue (surgical debridement)
• Penicillin G in high doses if more serious infection
Of poorly defined clinical value are:
• administration of antitoxin
• hyperbaric oxygen (dive chamber) adjunct therapy (??inhibit growth of anaerobe??)

21. Summary of C.
perfringens
Infections

22. Micro & Macroscopic C. perfringens
NOTE: Large rectangular NOTE: Double zone of hemolysis
gram-positive bacilli
Inner beta-hemolysis = θ toxin
Outer alpha-hemolysis = α toxin

23. Clostridium perfringens
• Pathogenesis: Traumatic open wounds or compound
fractures lead to muscle damages and contamination
with dirt etc,
• Mainly in war wounds, old age, low blood supply and
amputation of thigh (required prophylaxis with
penicillin
• Prevention and Treatment
• Remove dead tissue , debris and foreign bodies .Penicillin
and hyperbaric oxygen in some cases

24. Clostridium perfringens
• Can leads to the following diseases
• 1) Wound Contamination
• 2) Wound infection
• 3) Gas Gangrene - most important disease
• 4) Gas Gangrene of the uterus in criminal abortion
• 5) Food Poisoning : Spores are swallowed  Germinate
in gut after 18 hours(Toxin production) abdominal
pain and diarrhoea

25. Clostridial Cellulitis

26. C. perfringens Virulence Factors
Major
Minor

27. Exotoxins Associated with C.
perfringens Types A-E
Major

28. C. perfringens Nagler Reaction
NOTE: Lecithinase (α-toxin; phospholipase) hydrolyzes
phospholipids in egg-yolk agar around streak on right. Antibody
.against α-toxin inhibits activity around left streak

29. Clostridium tetani

30. Clostridium tetani — agent of tetanus
Morphology and Physiology-
• long thin gram-positive organism that stains gram negative in old cultures
• round terminal spore gives drumstick appearance
• motile by peritrichous flagella
• grow on blood agar or cooked meat medium with swarming
• beta-hemolysis exhibited by isolated colonies
• spores resist boiling for 20 minutes
Antigenic Structure-
flagella (H), somatic (0), and spore antigens. Single antigenic toxin characterizes all strains.
Pathogenicity Determinants"
• play a role in local infection only in conjunction with other bacteria that create suitable
environment for their invasion
• systemic-acting, plasmid-mediated A-B neurotoxin (tetanospasmin) produced intracellularly
 Mode of Action — one of most poisonous substances
• binds gangliosides in synaptic membranes (synapses of neuronal cells) and blocks
release of inhibitory neurotransmitters; continuous stimulation by excitatory transmitters
• muscle spasms (spastic paralysis) (trismus (lockjaw), risus sardonicus, opisthotonos),
cardiac arrhythmias, fluctuations in blood pressure
Lab Identification"
• use characteristics of resistance to heat, motility, and toxin production to help identify
Diagnosis/Treatment/Prevention
• empirical diagnosis on basis of clinical manifestations
• treat to prevent elaboration and absorption of toxin
 clean wound (debridement), control spasms
 metronidazole administered to eliminate vegetative bacteria that produce neurotoxin
 passive immunity (human tetanus immunoglobulin); vaccination (active) as preventative
 antitoxin administered to bind free tetanospasmin

31. C. tetani Infections

32. .( Summary of Clostridium tetani Infections (cont

33. Clostridium tetani Gram Stain
NOTE: Round terminal spores give cells a
.“drumstick” or “tennis racket” appearance

34. Clinical Forms of Tetanus

35. Opisthotonos in Tetanus Patient

36. Risus Sardonicus in Tetanus Patient

37. Clostridium botulinum

38. CLOSTRIDIUM BOTULINUIM
• Found in soil ponds and lakes
• Toxin is exotoxin (protein) heat labile at 100 OC and
resist gastrointestinal enzymes
• It is the most powerful toxin known Lethal dose 1 µg
human and 3 kg kill all population of the world .It
dictated for by lysogenic phage
• Botulism
• From canned food., sea food e_g. salmon when it is not
well cooked (Spores resist heat at 100 oC ) then
multiply and produce toxin

39. CLOSTRIDIUM BOTULINUIM
• Symptoms
• Abnormal eye movement as if cranial nerve affected when
bulbar area of the brain affected. Finally the patient might
develop respiratory and circulatory collapse
• Enfantile Botulism
• Ingestion of Spores  germination in the gut  Botulism .
• Botulism Patogenesis
• Attacks neuromuscular junctions and prevents release of
acetylcholine that can leads to paralysis

40. CLOSTRIDIUM BOTULINUIM
• Laboratory diagnosis
• Suspected food and the patient faeces culture or serum
toxin detection by mice inoculation after weeks 
paralysis and death
• Treatment
• Maily supportive and horse antitoxin in sever cases
• Prevention
• Adequate pressure cooking autoclaving and heating of
food for 10 minutes at 100 OC

41. Clostridium dificile

42. Clostridium Difficile
• Normal flora in gastroentestinal tract after exposure to
antibiotics and killing of other normal flora, this organism
will multiply witch then produce toxin that has two
components
– A–Subunit enterotoxin (cause diarrhea)
– B-Subunit Cytotoxic ( kill the cells ie necrosis)
– Hyaluronidase and spore forming so bacteria can survive for a
month in hospital environment
• PSEUDOMEMBRANE COLITIS is the clinical manifestation of
this disease which composed of bacteria , fibrin , WBCs and
dead tissue cells
• Sever dehydration , intestinal obstruction and perforation are
some of complication of this syndrome

43. Clostridium Difficile
Laboratory diagnosis: this organism hard to grow in the
laboratory required special media and growth of the
organism in solid media required cell line culture to
demonstrate cytotoxicity of the organism.
The simplest method for diagnosis by detection of the
toxin in the stool by immunological testing (ELISA)

44. Clostridium Difficile
• Treatment : Metronidazole or and oral vancomycin in
sever cases
• Prevention: This organism form spores and hard to
control in the hospital because they are resistant to
alcohol decontamination ( use Na hypochloride instead).
• Patient need to be isolated and contact need to be
screened to find out if they carrying the toxic strain of
the bacteria.