Laboratory diagnosis in infections

produced by anaerobic bacteria

*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)

(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

(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

(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

(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)

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

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

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)

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

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 (-)

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

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).

Genus Clostridium
- Clinical significance Exogenous infections:

Gas gangrene
Tetanus
Botulism
Food poisoning

Endogenous infections:
Clostridium difficile

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

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

Gas gangrene: Clostridium perfringens

Oedema, necrosis, large
blisters, crepitation
Lower image: large
incision for oxygen
exposure of infected
tissues

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)

Gas gangrene clostridia: Cl. perfringens

- Isolation and identification Blood agar: double
hemolysis

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)

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)

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)

The reverse CAMP test

Identification
of Clostridium
perfringens:
(A) Reverse CAMPpositive Clostridium
perfringens (bow tie)
(B) reverse CAMPnegative Clostridium septicum
streaked at right angles to
(C) Streptococcus
agalactiae (group B)

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

Obligate Anaerobic Bacteria

- Human infections Exogenous sporulating
Gram positive bacilli:
Genus Clostridium:

Endogenous nonsporulating
Gram negative bacilli:
Family Bacteroidaceae:
Bacteroides, Prevotella,
Prophyromonas,
Fusobacterium

Gas gangrene clostridia

Clostridium tetani
Clostridium botulinum
Clostridium difficile* (may
cause both exogenous and
endogenous infections)

Gram positive bacilli:

Propionibacterium acnes
Actinomyces israelii

Gram positive cocci:

Peptococcus,
Peptostreptococcus

Gram negative cocci:

Veillonella

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

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

Left: Risus sardonicus (rigid facial grin)

Right: Opisthotonos (spastic contraction with
hyperextension of head, neck and spine)

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

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-adolesc
ent.html#printable
http://www.nhs.uk/Conditions/vaccinations/Pages/vaccina
tion-schedule-age-checklist.aspx
http://en.pediatricblog.info/2011/02/romanian-mandatoryvaccination-schedule.html

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)
YESQ2: 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

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
YESQ2: 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)

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

Obligate Anaerobic Bacteria

- Human infections Exogenous sporulating
Gram positive bacilli:
Genus Clostridium:

Endogenous nonsporulating
Gram negative bacilli:
Family Bacteroidaceae:
Bacteroides, Prevotella,
Prophyromonas,
Fusobacterium

Gas gangrene clostridia

Clostridium tetani

Clostridium botulinum
Clostridium difficile* (may
cause both exogenous and
endogenous infections)

Gram positive bacilli:

Propionibacterium acnes
Actinomyces israelii

Gram positive cocci:

Peptococcus,
Peptostreptococcus

Gram negative cocci:

Veillonella

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

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!!

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

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

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; antibioticassociated 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)

Clostridium difficile
- Prevention guidelines in clinical settings Examples:
http
://www.documents.hps.scot.nhs.uk/about-hps/hpn/clostridiu
m-difficile-infection-guidelines.pd
f
http://www.idsociety.org/uploadedFiles/IDSA/GuidelinesPatient_Care/PDF_Library/cdiff2010a.pdf
http://d2j7fjepcxuj0a.cloudfront.net/wp-content/uploads
/2013/04/ACG_Guideline_Cdifficile_April_2013.pdf

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

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

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