Clostridium

Clostridium spp.
Gram positive
bacilli
no typical arrangements in smears
anaerobic

spore-forming
motile (C. perfringens: nonmotile )
ubiquitous (soil, intestinal tract of animals)
some species members of normal microbial flora
(GIT) in humans

Clostridium spp.: spores

different shapes: oval, round, kidney shaped,...
location: subterminal and terminal
typically: deform the bacterial cells
resistant to heat, light, drying and radiation
survive for decades
germination: anaerobic environment

Clostridium spp.
C. perfringens

C. difficile
C. botulinum

C. tetani

Clostridium perfringens

typical representative of the genus

subterminal ovoid spore
nonmotile
generation time: 8-10 minutes
wide distribution in nature (soil, decaying
vegetation, intestinal tract of animals)
spores frequently present (transient) in human
GIT

C. perfringens spores

spore

spore

C. perfringens
Virulence factors:
spore
polysaccharide capsule; protection from phagocytosis
numerous toxins and enzymes (~ 20):
major (lethal toxins) and minor virulence factors
rapid multiplication

C. perfringens
Major virulence factors (lethal toxins)
(alpha) toxin

mechanism: phospholipase C
target cells: erithrocytes, leukocytes,
endothelial cells; epithelial cells
effects: necrosis; increased vascular
permeability

(beta) toxin

mechanism: pore forming protein; K+

efflux from cells and Ca2+, Na+, and
Cl- influxes
target cells: leukocytes, endothelial
cells
effects: necrosis; increased vascular
permeability

C. perfringens
Major virulence factors (lethal toxins)
(epsilon) toxin mechanism: unknown; acts from the
surface of cells and causes K+ efflux
target cells: epithelial and endothelial
cells
effects: edema; increased vascular
permeability; vascular damage

(iota) toxin

mechanism: A-B toxin; A subunit ADP

ribosylation of cytoskeleton proteins
target cells: muscle cells; ?
effects: necrosis; dermonecrosis

C. perfringens
Major virulence factors: 5 biotypes
Toxin

Type (alpha)
A
B
C
D
E

+
+
+
+
+

(beta)
+
+
-

(epsilon)

(iota)

+
+
-

Type A: majority of infections in humans

C. perfringens
Major /minor (?) virulence factor: enterotoxin
Clostridium perfringens enterotoxin (CPE)
single polypeptide (35 kDa)
binding of toxin to receptor
formation of small
complex (90 kDa)
rapid oligomerisation into a
hexamer of 450 kDa
a pore through which
calcium gains entry to cytoplasm
altered cell
membrane permeability
Also: disruption of tight junctions
allows CPE access
to basolateral membrane
formation of additional
hexamer complexes
death of enterocytes + increased paracellular permeability:
diarrhea

C. perfringens
Major /minor (?) virulence factor: enterotoxin

C. perfringens
Minor virulence factors
(delta) toxin

hemolysine

(theta) toxin

pore forming hemolysine; cytolytic;

increased vascular permeability

(kappa) toxin

collagenase and gelatinase

(lambda) toxin

protease

(mu) toxin

hyaluronidase

(nu) toxin

DN-ase; hemolysine

neuraminidase

exposure of receptors

C. perfringens
Diseases (and epidemiology)
I Soft tissue infections
C. perfringens + other histotoxic clostridia
(C. septicum, C. sordellii, C. histolyticum, C. novyi)
exogenous or endogenous exposure to spores
- clostridial myonecrosis or gas gangrene
- anaerobic cellulitis
- anaerobic fasciitis
- endometritis

C. perfringens
Diseases (and epidemiology)
at the site of trauma or a
recent surgical wound; at the
site of compromised
circulation
extensive tissue damage +
gas + (sepsis)

gas gangrene

C. perfringens
Diseases (and epidemiology)

II Clostridial food poisoning

enterotoxin-producing C. perfringens type A
abdominal cramps and diarrhea 8-16 hours after
eating contaminated food (spores frequently present
in meat, meat products)
among the most common causes of foodborne
illnesses in developed countries
spores survive high temperatures during
initial cooking
germination during cooling
vegetative forms multiply at room temperature
ingestion of vegetative forms (if served without
adequate reheating)
production of enterotoxin

C. perfringens
Diseases Diseases
(and epidemiology)
III Enteritis necroticans (pig-bel)
beta toxin-producing C. perfringens type C
(pore-forming toxin; tripsin sensitive)
necrotizing infection of jejunum and ileum
occurs sporadically in parts of Asia, Africa, and
South Pacific
after meat feasts: primarily affects children with
severe protein malnutrition and on diet containing
trypsin inhibitors (sweet potato) + Ascaris
coinfection

C. perfringens
Laboratory diagnostics
specific techniques for cultivation of anaerobic bacteria
soft tissue infections: no cells in direct smears

food poisoning:
- significant finding:
105 bacteria/g of food or 106 bacteria/g feces
- detection of enterotoxin in stool: Ag-Ab assays

C. perfringens
Prevention
Soft tissue infections: surgery + antibiotics
Clostridial food poisoning:

Enteritis necroticans: vaccine (toxoid of beta toxin C.

perfringens type C)

Clostridium difficile

typical representative of the genus

subterminal ovoid spore
motile
may be present as a member of normal microbial
flora (colon)

spore

C. difficile
Medical importance
causes gastrointestinal disease in individuals
undergoing antibiotic therapy

leading cause of hospital-acquired diarrhea:

C. difficile-associated disease (CDAD)

CDAD: mild diarrhea to pseudomembranous colitis

C. difficile
Virulence factors
spore

capsule polysaccharide; protection from

phagocytosis
motility
hidrolytic enzymes connective tissue degradation

toxin A and toxin B

C. difficile
Virulence factors
toxin A
(TcdA)

toxin B
(TcdB)

large proteins
internalization: receptor-mediated endocytosis
require an acidic endosome for translocation into cytosol
glucosyltransferases that inactivate Rho proteins

Rho proteins small GTPases; primary regulators of actin

cytoskeleton
Effect on cells: loss of structural integrity; rearranged microvilli;
cell rounding; cell death
disorganization of cell cytoskeleton
cell death

C. difficile
CDAD
pathogenesis

C. difficile
CDAD: risk factors
1. disruption of normal microbial flora of colon through
exposure to antibiotics
nearly all antimicrobials have been implicated in development
of CDAD; greater risk: cephalosporins, clindamycin,
fluoroquinolones
2. hospitalization (long-term care facility residence)

advanced age, severe underlying disease,

immunocompromising conditions, gastrointestinal surgery,
gastric acid suppression

C. difficile
Epidemiology
up to 60% of healthy adults have detectable serum IgG
and IgA against TcdA and TcdB
community sources: soil, water, pets, meat, vegetables
C. difficile colonizes 60 70% of healthy newborns and
infants up to 12 18 months of age when normal colonic
microflora is established
significant decrease in
colonization rate
about 3% of healthy adults in general population are
colonized with C. difficile vs. 20 - 40% of hospitalized
patients

C. difficile
Epidemiology
most commonly a nosocomial pathogen
reservoirs: patients, healthcare workers, contaminated
hospital environment
acquisition of C. difficile: oral ingestion of spores

incidence and severity of CDAD have increased dramatically

over the last decade: emergence and global dissemination of
hypervirulent epidemic strain of C. difficile BI/NAP1/027
(increased production of toxin A and toxin B; additional binary
toxin)
US: > 250,000 CDAD cases per year; total additional health
care costs approaching 1 billion US$ annually
community-acquired CDADs have also been described

C. difficile
Laboratory diagnostics
Specific toxin identification is required for definitive
diagnosis
Ag-Ab assays for detection of toxin A and toxin B in stool
samples

Culture: rarely

C. difficile
Prevention
appropriate use of antimicrobial therapy

isolation of suspected or infected patients

hand hygiene with chlorhexidine - containing soap (not
ethanol solutions because these do not kill spores) and
wearing protective gowns by all healthcare providers after
every contact with a patient with suspected or known CDAD
patient-dedicated noncritical equipment

environmental cleaning: chlorine-containing agents

vaccine: in development

C. botulinum strains - general

properties
typical representative of the genus
subterminal oval spore
motile
ubiquitous
produce BoNT
Defining C. botulinum on the basis of BoNT production
has resulted in the species encompassing a range of
very diverse bacteria.

I Phenotypic classification of C. botulinum strains in

accordance with antigenic specificity of the
neurotoxin (7 antigenically distinct variants)
A, B, C (C1 i C2), D, E, F, G
II Phenotypic classification of C. botulinum strains
in accordance with metabolic properties and toxin
types
4 groups
III Genotypic classification of C. botulinum strains
at least 4 genomic species
there is a need for a major
change in taxonomy

BoNT: structure and synthesis

produced as progenitor toxin complex:
BoNT with associated proteins:
hemagglutinin proteins (HA)
non-toxic non-hemagglutinin protein (NTNH)
role of associated proteins:
- increase stability of BoNT in GIT

- increase potency of BoNT (enable

transfer through intestinal mucous
membrane)

Non-toxic associated proteins

Non-toxic non-hemagglutinin
(NTNH)

Hemagglutinins (HA)

BoNT: structure
A-B toxin:
light chain [L] (50 kDa; 1 - 448 amino acids)
heavy chain [H] (100 kDa; 449 - 1280 amino acids)
linked by a disulphide bridge

BoNT: specific affinity for

neuromuscular junction

BoNT: mechanism of action

BoNT acts by binding presynaptically to
high-affinity recognition sites on the
cholinergic nerve terminals and
decreasing the release of acetylcholine,
causing a neuromuscular blocking
effect.

BoNT: receptors
absorption of toxin into the bloodstream
binds irreversibly to a toxin receptor on the
nerve cell membrane at the neuromuscular
junction

receptor for
BoNTB

receptor-mediated andocytosis
require an acidic endosome for
translocation into cytosol
THE POINT OF NO
RETURN: once
endocytosed, the
toxin can no longer
be neutralized

receptor for
BoNTA

BoNT: mechanism of action

light chain: Zn-dependent protease

proteolytic cleavage of SNARE proteins:

synaptobrevin, syntaxin, SNAP 25

Acetylcholine is stored in the synaptic vesicles; released

into the synaptic cleft by fusion with the presynaptic
membrane, through the process of exocytosis (consequence
of an influx of calcium ions that is triggered by a nerve action
potential)
Calcium-regulated exocytosis requires the actions of
proteins located on the vesicles in the cytosol and on the
presynaptic membrane
SNARE: protein components of the neuroexocytosis
apparatus
release of the vesicle from the cytoskeleton
protein on the
synaptic vesicle and proteins on the presynaptic membrane
act as anchors that pull the membranes together
fusion
aaaaa
and exocytosis

Botulism
food borne botulism
caused by ingestion of preformed toxin
home-canned foods (spores survive an inadequate
cooking and canning process
germination)
onset: 18 to 36 hours after exposure (6 hours to 8 days)
initial symptoms: gastrointestinal
neurologic symptoms: symmetric descending flaccid
paralysis (cranial nerves, upper extremities, respiratory
muscles, lower extremities)

Botulism
infant botulism
C. botulinum spores enter and colonize the gastrointestinal
tract and produce toxin
spectrum of disease: from mild constipation to sudden
death

wound botulism

occurs when anaerobic conditions within a wound allow

germination of C. botulinum spores
clinical manifestations are similar to those seen in
foodborne botulism (gastrointestinal symptoms absent)
black tar heroin
Source: drug, a cut in the drug, dirty
injection equipment, or contamination
during the preparation process.

BoNT: botulizam
adult infectious botulism
result of intestinal colonization with C. botulinum and in
vivo toxin production
patients have a history of abdominal surgery,
gastrointestinal tract abnormalities, or recent antibiotic
treatment (disruption in normal microbial flora)

C. botulinum
Epidemiology
ubiquitous in soil
critical factor: contamination of food
home canned products that have not been heated at
temperatures sufficient to kill C. botulinum spores
alkaline conditions provided by foods (vegetables mushrooms, grean beans) support the growth of C.
botulinum
acidic conditions provided by foods (canned fruits) do not
support the growth of C. botulinum

inadequately sterilized commercial products have also been

implicated
germination of spores is favored in food kept at warm
temperatures under anaerobic conditions for a long period of
time

C. botulinum
Laboratory diagnostics
Culture of bacteria (stool, wound swab, food) and detection
of toxin (serum, stool, food)

Rapid diagnostics
Detection of bacteria:
molecular techniques
Detection of toxin:
Ag-Ab assays

Botulism
Prevention
Adequate pressure cooking or autoclaving in the canning
process kills spores.
Heating food at 80C for 30 minutes or at 100C for 10
minutes before eating destroys toxin.
Food from damaged cans or those with evident positive
pressure shoud be discarded.
Honey is not recommended for infants under 1 year of age.

BoNT as a biological
warfare agent
listed as Category A agent by CDC
the most toxic substance known
1 gram of crystalline toxin can kill >1
million people if dispersed and inhaled
evenly
extreme potency and lethality; ease of
production and transport; need for
prolonged intensive care

BoNT as a therapeutical agent

first bacterial toxin used for treatment of
human disease
manufactured for medical use in 1989
under name Oculinum (BoNT A)
licensed for treatment of strabismus and
blepharospasm (eye conditions
characterized by excessive muscle
contraction)

Blepharospasm treated with Oculinum

Vangelova, Luba. Botulinum Toxin: A Poison that Can Heal. Available at:
http://www.fda.gov/fdac/features/095_bot.html.

Medical uses of BoNT

now manufactured under the name Botox
used for treating migraine headaches, chronic
low back pain, stroke, dystonias (neurologic
diseases involving abnormal muscle posture
and tension)
frequent injections allows an individual to
develop antibodies
studies carried out to
determine feasibility of other types of BoNT
BoNT B manufactured for treatment of cervical
dystonia in 2000 as Myobloc

Cosmetic use of BoNT

Botox approved for Cosmetic use in April, 2002
Myobloc not approved for cosmetic use (used
in many cosmetic procedures anyway)

BoNT A (Botox )
Botox injection patient 13 weeks after
injection

Sadick, N. and A.R. Herman (2003). Comparison of Botulinum Toxins A and B in the
Aesthetic Treatment of Facial Rhytides. Dermatologic Surgery 29:340-347.

BoNT B (Myobloc)
Myobloc injection patient 11 weeks after
procedure

Sadick, N. and A.R. Herman (2003). Comparison of Botulinum Toxins A and B in the
Aesthetic Treatment of Facial Rhytides. Dermatologic Surgery 29:340-347.

however,

Clostridium tetani general properties

typical representative of the genus
round terminal spore
motile
ubiquitous (soil)

spore

drumstick
appearance
spore

C. tetani: virulence factors

noninvasive bacterium
tetanospasmin neurotoxic exotoxin
antigenic specificity of neurotoxin: identical in all
strains
A-B toxin

Tetanospasmin: mechanism of
action
local multiplication of C. tetani and production od
tetanospasmin
tetanospasmin enters the presynaptic terminals
of lower motor neurons
retrograde axonal
transport system in nerves
reaches CNS
and acts at the level of the anterior horn cells
(spinal cord)
blocks postsynaptic inhibition of
spinal motor reflexes
spasmodic
contractions of muscles

Tetanospasmin: mechanism of
action
neurotransmission is controlled by the balance
between excitatory and inhibitory neurotransmitters
(gamma-aminobutyric acid, glycine)
inhibitory neurotransmitters prevent depolarization of
the postsynaptic membrane and conduction of the
electrical signal
in the absence of inhibitory neurotransmitters:
uncontrolled excitation
tetanospasmin interferes with release of GABA and
glycine (presynaptic activity).

Tetanospasmin: mechanism of action

light chain: Zn-dependent protease

proteolytic cleavage of a SNARE protein:

synaptobrevin

Tetanospasmin: mechanism of action

C. tetani: disease
Tetanus
C. tetani spores introduced into wounds
contaminated with soil or foreign bodies (low
oxidation-reduction potential)
local
multiplication and toxin production
neurotoxicity
muscle spasms

Tetanus
- generalized tetanus
incubation period: few days to several weeks (the more
peripheral the wound, the longer the incubation time)

initial symptom: cramping and twitching of muscles around

a wound
descending spastic paralyses
Complications: fractures, intramuscular hematoma,
muscle ruptures, pulmonary, renal, and cardiac problems

Tetanus - trismus

Tetanus - opisthotonos

Tetanus - opisthotonos

Tetanus
- Local tetanus
toxin travels along the neural route (peripheral nerves)
ascending tetanus
disease confined to the extremities
may last for months but usually resolves spontaneously

-Tetanus neonatorum
contamination of umbilical stump
generalized form
highest mortality

Tetanus neonatorum - opisthotonos

C. tetani
Epidemiology
ubiquitous in soil
can be found in the gastrointestinal flora of humans, horses,
and other animals
One million cases of tetanus occur annually in the world.

US: one case per million per year

Tetanus
Prevention

DTP vaccine diphtheria tetanus pertussis

DT vaccine diphtheria - tetanus
Vaccine: toxoid/anatoxin of tetanospasmin
With adequate immunization
tetanus is a completely
preventable disease!