This document discusses laboratory diagnosis of infections caused by obligate anaerobic bacteria. It defines various types of bacteria based on their ability to grow with or without oxygen. Obligate anaerobes cannot grow in the presence of oxygen because they lack enzymes like superoxide dismutase and catalase to break down harmful oxygen byproducts. Specimen collection and transport methods aim to maintain an oxygen-free environment. Identification techniques for certain pathogenic anaerobes that cause infections like gas gangrene, tetanus, and botulism are also outlined.

1. Laboratory diagnosis in infections
produced by anaerobic bacteria

2. *Definition of terms:
bacterial growth in relation with respiratory
processes (use of O2, CO2)
• Obligate aerobes need oxygen because they cannot
ferment or respire anaerobically (e.g. Mycobacterium
tuberculosis)
• Obligate anaerobes are poisoned by oxygen
• Facultative anaerobes can grow with or without oxygen
(e.g. Staphylococcus, Streptococcus, E.coli)
• Microaerophiles need some amount of oxygen but are
poisoned by high concentrations of oxygen (e.g.
Campylobacter, Helicobacter, Neisseria gonorrhoeae)

3. (Obligate) Anaerobic bacteria
- general aspects & definition of terms -
• Energy generated exclusively by anaerobic fermentation
(does not generate superoxide radicals = O2 anions)
• Can only grow in the total absence of O2;
• WHY?: Anaerobic bacteria lack the enzymes:
– Superoxide dismutase (SOD)
– Catalase

4. (Obligate) Anaerobic bacteria
- general aspects & definition of terms - continued
• Superoxide dismutases - antioxidant factors; enzymes
which catalyze the dismutation (partitioning) of the
superoxide radical (O2 anion) into:
– Molecular O2 or
– Hydrogen peroxide (H2O2)
• Superoxide = by-product of O2 metabolism; high cellular
toxicity
• Hydrogen peroxide = less toxic; degraded by catalase

5. (Obligate) Anaerobic bacteria
- general aspects & definition of terms - continued
CONCLUSIONS:
• fermentation in the presence of O2 → superoxide
radicals – toxic if not dismuted (partitioned) by superoxid
dismutase into O2 / H2O2;
• furthermore H2O2 – toxic if not decomposed by catalase
• Anaerobic bacteria lack both enzymes (superoxid
dismutase and catalase) → mandatory absence of O2 in
order for anaerobic bacteria to avoid toxic effects of
superoxide radicals and/or H2O2

6. (Obligate) Anaerobic bacteria
- general aspects & definition of terms -
”Friends or Foes?”
• Colonize the human body
• involved in the balance of the normal microbial flora:
skin, oropharynx, gastro-intestinal tract, uro-genital tract
(urethra, vagina)
• Cause severe infections (endogenous and exogenous)

7. Obligate Anaerobic Bacteria
- Collection and transport of specimens -
• Inoculation asap (within 10 minutes) due to toxicity of
atmospheric O2
• Transport: anaerobic tubes with transportation media
e.g. modified Cary Blair, Stuart
– minimal nutrients to increase survival of organisms without
multiplication
– sodium thioglycollate - to provide low oxidation-reduction
potential
– alkaline pH – to minimize bacterial destruction by acid
production
– phenol red indicator (red at alkaline pH, yellow at acidic pH)
– Redox indicator: resazurin – turns pink in the presence of O2

8. Obligate Anaerobic Bacteria
- Collection and transport of specimens -
continued
“Hungate tubes”:
• Disposable/autoclavable
screw thread style tube
designed to maintain
anaerobic culture
conditions
• butyl rubber stoppers,
• screw cap 9 mm opening

9. Obligate Anaerobic Bacteria
- Macroscopic and microscopic exam -
• Suggestive signs of anaerobic
infection:
– Fetid odour
– Purulent aspect
– Necrotic tissues
– Gas (e.g. ”gas gangrene” –
wound infected by Clostridium
perfringens)
• Microscopy: Gram stained
smears (methanol fixation to
preserve cellular elements)

10. Obligate Anaerobic Bacteria
- Isolation -
• Nonselective media: blood
agar
• Selective media requirements
(CLSI: Clinical Laboratory
Standards Institute):
– 3 days of strict anaerobic
incubation → mandatory
growth of:
– Bacterioides fragilis,
Clostridium perfringens +
Fusobacterium nucleatum,
Peptostreptococcus
anaerobius, Bacteroides levii

11. Obligate Anaerobic Bacteria
- Identification -
• API 20 A
Identification of anaerobes in 24-48 hours
• Fermentation tests, which are the reference tests for the
identification of anaerobes
• Easy-to-use: suspension prepared directly in the API 20
A medium.
• Polyvalent system for all anaerobes, both Gram (+) and
Gram (-)

12. Obligate Anaerobic Bacteria
- Human infections -
Exogenous sporulating
• Gram positive bacilli:
• Genus Clostridium:
– Gas gangrene clostridia
– Clostridium tetani
– Clostridium botulinum
– Clostridium difficile* (may
cause both exogenous and
endogenous infections)
Endogenous nonsporulating
• Gram negative bacilli:
– Family Bacteroidaceae:
• Bacteroides, Prevotella,
Prophyromonas,
Fusobacterium
• Gram positive bacilli:
– Propionibacterium acnes
– Actinomyces israelii
• Gram positive cocci:
– Peptococcus,
Peptostreptococcus
• Gram negative cocci:
– Veillonella

13. Bacterial survival outside
host
Spores: reproductive structures adapted
for longtime survival in unfavourable
conditions
(etymology: ancient Greek spora = seed)
Bacterial spores - outer layer of keratin resistant to chemicals, staining and
heat → bacterium able to stay dormant for years, protected from
temperature differences, absence of air, water and nutrients
Spore forming bacteria:
• Genus Clostridium;
• Bacillus spp (B. anthracis).

14. Genus Clostridium
- Clinical significance -
• Exogenous infections:
– Gas gangrene
– Tetanus
– Botulism
– Food poisoning
• Endogenous infections:
• Clostridium difficile

15. Definition of terms: Gangrene
• tisular death (necrosis) caused by lack of blood supply
(= absence of O2 and nutrients in the respective area of the
organism)
• potentially life-threatening condition
• may occur by:
– injuries, trauma (compression of blood vessels)
– frostbite (freezing of exposed extremities)
– infection
– chronic diseases affecting blood circulation e.g. diabetes

16. Gas gangrene clostridia
• Gas gangrene – severe invasive
infection starting from infected
wounds, rapid systemic invasion
• Caused by: Clostridium
perfringens, + Cl. oedematiens,
Cl. histolyticum – found in water,
soil air + intestinal comensals
• Severity augmented by toxin
production
• Collection of specimens: profound
wound secretion, tissue fragments

17. Gas gangrene: Clostridium perfringens
• Oedema, necrosis, large
blisters, crepitation
• Lower image: large
incision for oxygen
exposure of infected
tissues

18. Gas gangrene clostridia
- Microscopic examination -
Gram stained smear:
• total absence of cells (no
PMNs, no epithelial cells,
etc)
• short, thick, Gram
positive bacilli
• no spores (no spore
forming in vivo)

19. Gas gangrene clostridia: Cl. perfringens
- Isolation and identification -
Blood agar: double
hemolysis

20. Gas gangrene clostridia: Cl. perfringens
- Isolation and identification - continued
Egg yolk agar (EYA): enriched
medium for presumptive id of
anaerobes e.g. Clostridium
• Egg yolk suspension: detection
of enzymes:
– lipase (iridescent sheen on
colony surface) and
– lecitinase (opaque precipitate
around colonies)

21. Gas gangrene clostridia: Cl. perfringens
- Isolation and identification - continued
• Reverse CAMP test
Principle: synergistic effect between Streptococcus
agalactiae (group B) and hemolytic Clostridium
perfringens
• Initially intended as an improvement of CAMP test i.e.
replacement of S.aureus by Clostridium perfringens for
the identification of Streptococcus agalactiae (Group B)
• Then the idea comes up to use Streptococcus
agalactiae (group B) to identify Clostridium perfringens
(= the reverse CAMP test)

22. The CAMP test:
id of Streptococcus agalactiae
(A) Streptococcus (group
B) positive test
(enhanced hemolysis)
(B) Streptococcus
pyogenes (group A)
negative test
(C) Staphylococcus
aureus – replaced by
Clostridium perfringens
in reverse CAMP test
(see next slide)

23. The reverse CAMP test
Identification
of Clostridium
perfringens:
(A) Reverse CAMP-
positive Clostridium
perfringens (”bow tie”)
(B) reverse CAMP-
negative Clostridium septicum
streaked at right angles to
(C) Streptococcus
agalactiae (group B)

24. Clostridium perfringens
- Antimicrobial susceptibility -
• Sensitivity to: penicillin G, erythromycin, ampicillin,
metronidazole
• Natural resistance to tetracyclines

25. Obligate Anaerobic Bacteria
- Human infections -
Exogenous sporulating
• Gram positive bacilli:
• Genus Clostridium:
– Gas gangrene clostridia
– Clostridium tetani
– Clostridium botulinum
– Clostridium difficile* (may
cause both exogenous and
endogenous infections)
Endogenous nonsporulating
• Gram negative bacilli:
– Family Bacteroidaceae:
• Bacteroides, Prevotella,
Prophyromonas,
Fusobacterium
• Gram positive bacilli:
– Propionibacterium acnes
– Actinomyces israelii
• Gram positive cocci:
– Peptococcus,
Peptostreptococcus
• Gram negative cocci:
– Veillonella

26. Clostridium tetani
• Habitat: intestinal tract of animals (sheep, cattle);
vegetative bacteria eliminated with faceces;
contamination of soil (spore formation)
• Infection occurs via:
– Skin lesions contaminated with spores e.g. wound highly
contaminated with dirt, dust; extensive wounds with crushed
tissues and foreign bodies (accidents);
– spores germinate into vegetative bacteria which multiply at the
entry and produce tetanic toxin (disseminated) – 2 components:
• Tetanospasmin – muscle spasms
• Tetanolysin – cardiotoxic

27. Clostridium tetani
Clinical significance:
• Tetanus = Generalized tetanus (most comon form):
– onset with trismus (spasms of the face and chewing muscles
popularly called “lockjaw”→ characteristic facial expression risus
sardonicus or sardonic grin);
– further evolution: swallowing becomes increasingly difficult;
severe spastic hyperextension of head, neck and spine
(opisthotonos)
(effects of the tetanus exotoxin: tetanospasmin)
• Lethal outcome ~ 1 in 10 cases - spastic paralysis of
respiratory muscles

28. Left: Risus sardonicus (rigid facial grin)
Right: Opisthotonos (spastic contraction with
hyperextension of head, neck and spine)

29. Tetanus
• Vaccine preventable disease: several vaccines used to
prevent tetanus among children, adolescents, and
adults; e.g. combined vaccines against diphteria,
pertusis and tetanus (e.g. DTaP) or tetanus and diphteria
(e.g.TD) – Immunization schedules
• In Romania: tetanus containing vaccines given at the
ages of:
– 2, 4, 6, 13 months, 4 years (DTP) + 14 years (dT) +
– (recommended) dT every 10 years

30. Definition of terms: Immunization schedule
• series of vaccinations, including the timing of all doses,
which may be either recommended or compulsory,
depending on the country of residence
• Examples:
• http://www.cdc.gov/vaccines/schedules/hcp/child-adolescent.h
• http://www.nhs.uk/Conditions/vaccinations/Pages/vaccination-
• http://en.pediatricblog.info/2011/02/romanian-mandatory-vacc

31. Tetanus prophylaxix in routine wound
management
1st
step: Assess wound - Clean, minor wound:
• Q1: Has patient completed a primary tetanus-diphteria
series? (= minimum 3 doses of tetanus- and diphteria
containing vaccine: e.g. at 2, 4 and 6 months of age)
– NO/Unknown: Administer vaccine today (i.e. complete series per
age-appropriate vaccine schedule)
– YES→Q2: Was the most recent dose within the past 10 years?
• NO: Administer vaccine today (next dose per age-appropriate
schedule)
• YES: vaccine not needed today; next dose will be given at 10 years
after the last dose

32. Tetanus prophylaxix in routine wound
management - continued
1st
step: Assess wound: “tetanigenic potential”:
contaminated with dirt, faeces, saliva, soil; puncture
wounds (lack of O2 in profound layers of wound); animal
bites, burns, frostbite
• Q1: Has patient completed a primary tetanus-diphteria
series?
– NO/Unknown: vaccine + tetanus immune globulin (TIG) today
– YES→Q2: Was the most recent dose within the past 5 years?
• NO: Administer vaccine today (next dose per age-appropriate
schedule)
• YES: Vaccine not needed today (next dose at 10 years from last
dose)

33. Clostridium tetani
• Laboratory diagnosis only required in suspicion of
iatrogenic infections e.g. infection of umbilical cord
stump, post-partum infections, etc
• In most cases diagnosis relies on clinical aspect and
history (tetanigenic circumstances e.g. wounds
contaminated with dirt, faeces, saliva, soil; puncture
wounds; animal bites, burns, frostbite)
• IMPORTANT FACTS:
– no human to human transmission
– Vaccine preventable

34. Obligate Anaerobic Bacteria
- Human infections -
Exogenous sporulating
• Gram positive bacilli:
• Genus Clostridium:
– Gas gangrene clostridia
– Clostridium tetani
– Clostridium botulinum
– Clostridium difficile* (may
cause both exogenous and
endogenous infections)
Endogenous nonsporulating
• Gram negative bacilli:
– Family Bacteroidaceae:
• Bacteroides, Prevotella,
Prophyromonas,
Fusobacterium
• Gram positive bacilli:
– Propionibacterium acnes
– Actinomyces israelii
• Gram positive cocci:
– Peptococcus,
Peptostreptococcus
• Gram negative cocci:
– Veillonella

35. Clostridium botulinum
- Clinical significance -
Foodborne Botulism:
• Ingestion of botulinic toxin
contained in canned
vegetables, fish, meat (toxin
production generated gas →
can lid appears
bulging/swollen)
• Toxin resists gastric acidity,
enters the intestine → lymph
vessels → blood stream→ acts
on nervous system:
progressive flaccid paralysis
Wound botulism: i.v. /
intradermic drug users
• Possible use as biological
weapon

36. Clostridium botulinum
- Clinical significance - continued
• Onset symptoms: double
vision, blurred vision,
drooping eyelids, slurred
speech, difficulty
swallowing, dry mouth,
and muscle weakness
• if untreated: descending
flaccid paralysis; death
by paralysis of respiratory
muscles
• Treatment: botulinum
antitoxin – asap!!

37. Clostridium botulinum
- Laboratory diagnosis -
• Performed in reference
laboratories
• Specimens: blood for
serology, vomit, faeces,
suspected food
• Identification based on:
– Gram stain: Gram positive
bacilli + spores;
– anaerobic growth;
– neurotoxin detection:
experimental disease in
mice; immunoassay;
molecular techniques

38. Obligate Anaerobic Bacteria
- Human infections -
Exogenous sporulating
• Gram positive bacilli:
• Genus Clostridium:
– Gas gangrene clostridia
– Clostridium tetani
– Clostridium botulinum
– Clostridium difficile* (may
cause both exogenous and
endogenous infections)
Endogenous nonsporulating
• Gram negative bacilli:
– Family Bacteroidaceae:
• Bacteroides, Prevotella,
Prophyromonas,
Fusobacterium
• Gram positive bacilli:
– Propionibacterium acnes
– Actinomyces israelii
• Gram positive cocci:
– Peptococcus,
Peptostreptococcus
• Gram negative cocci:
– Veillonella

39. Clostridium difficile
- Clinical significance -
• Pseudomembranous colitis: bloating and severe diarrhoea
• Endogenous: bacteria replaces normal intestinal flora that has
been compromised, usually following antibiotic treatment for an
unrelated infection; C. difficile gains a growth advantage (positive
selection) and overruns the intestinal microbiome; “antibiotic-
associated diarrhoea”
• Exogenous: accidental ingestion of spores e.g.
incomplete/incorrect hospital management of infected patient
(isolation, disinfection, etc) leads to spore contamination of objects
→ spore ingested by another patient (when prevention guidelines
are not strictly followed e.g. hand washing, cleaning, PPE,
disinfection)

40. Clostridium difficile
- Prevention guidelines in clinical settings -
Examples:
• http
://www.documents.hps.scot.nhs.uk/about-hps/hpn/clostridium
f
• http://www.idsociety.org/uploadedFiles/IDSA/Guidelines-Patie
• http://d2j7fjepcxuj0a.cloudfront.net/wp-content/uploads/2013/0

41. Obligate Anaerobic Bacteria
- Human infections -
Exogenous sporulating
• Gram positive bacilli:
• Genus Clostridium:
– Gas gangrene clostridia
– Clostridium tetani
– Clostridium botulinum
– Clostridium difficile* (may
cause both exogenous and
endogenous infections)
Endogenous nonsporulating
• Gram negative bacilli:
– Family Bacteroidaceae:
• Bacteroides, Prevotella,
Prophyromonas,
Fusobacterium
• Gram positive bacilli:
– Propionibacterium acnes
– Actinomyces israelii
• Gram positive cocci:
– Peptococcus,
Peptostreptococcus
• Gram negative cocci:
– Veillonella

42. Endogenous nonsporulating bacilli
Gram positive
• Propionibacterium acnes:
involved in juvenile acne,
blepharitis together with
staphylococci,
corynebacteria;
morphology similar to
corynebacteria
• Actinomyces israelii:
comensal flora of the oral
cavity; involved in
periodontal disease,
abscesses (in immune
compromised patients)
Gram negative
Bacteroides, Prevotella,
Prophyromonas,
Fusobacterium
- Normal flora
- Isolation in naturally
sterile sites – always
pathological

43. Endogenous nonsporulating cocci
Gram positive
• Peptococcus,
Peptostreptococcus –
normal oral flora; may be
involved in infections
together with other
anaerobes (e.g. skin
infections after human
bites)
Gram negative
• Veillonella – normal oral
flora; may be involved in
purulent alveolar
infections