This document outlines a presentation on the genus Clostridium. It begins with an introduction to Clostridium, noting that they are gram-positive, obligate anaerobic rods that can form endospores. It then covers the taxonomy and classification of Clostridium. The document discusses several important pathogenic Clostridium species - C. botulinum, C. perfringens, C. tetani, C. difficile. For each species, it covers transmission, clinical presentation, treatment and prevention. The final section discusses various diagnostic methods for Clostridium such as culture methods, gram staining, and molecular detection techniques.

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ClOSTRIDIA
ASHUTOSH KUMAR
MAHAFIZUR RAHMAN
ASHIK TILMILSINA
Outlines of presentation
1.What is clostridium
2.Taxonomy
3.Transmission
4.Clinical Presentation
5.Treatment and Prevention
6.Diagnostic Method
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What is clostridium ?
Genus of
1.Gram positive
3. Endospores
2.Obligate anaerobes
4.Rod shaped
Scientific Classification
Domain :- Bacteria
Kingdom :- Eubacteria
Phylum :-Firmicutes
Class :-Clostridium
Order :-Clostridiales
Family :- Clostridiaceae
Genus :-Clostridium
Species :- >100
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Important Pathogenic Species of Clostridium
Clostridium
botulinum
Clostridium
perifringers
Clostridium
Clostridium difficile
Clostridium tetani
Clostridium botulinum
First recognized and isolated in 1896 by
Van Ermengem
1. Gram positive rods
2. Obligate anaerobes :- oxygen is
poisonous to it but it can tolerate a
little amount of it because of
presence of superoxidase
dismutase(SOD) enzymes which is
a antioxidant defense present in
some cells
3. Can produce endospores only in
absence of oxygen
4. Able to produces neurotoxins during
the formation of spores
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Transmission
Clostridium botulinum is a soil bacterium and its spores are found in soil.I
It can easily transmitted in humans by
soil contaminated foods.
most common human diseases:
1. Food poisoning
2. Wound botulinum
3. Infant botulism
The spores of C.botulinum are heat resistant,
they can not be killed by simple boiling
Clostridium perfringens
Clostridium perfringens are:
1. Strongly Gram positive rods
2. Non motile
3. Short, thick, straight parallel sides with round
edges
4. Spore forming
Sporulation favored by alkaline environment and
absence of fermentable CHO
If spores are produced, they are large, oval and
central
Capsulated especially at the site of infection
Capsule disappears when organism is grown on
culture
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Transmission
C. perfringens is present in the
environment and can be
found in
- decaying vegetation
- marine sediments
- intestinal tract of humans
- meats and poultry
- soil
The most common diseases are
1.Food poisoning
2.Gas gangrene
Despite its potential danger C. perfringens is used as the
leavening agent in salt rising bread. The baking process is thought
to reduce the bacterial contamination, preventing negative effects
Clostridium difficile
It was first described in 1935 by Hall and O’ Toole
and then later in year 1970 recognized as cause of
antimicrobial associated diarrhea
Clostridium difficile is a Gram positive, rod shaped,
spore forming, anaerobic bacterium which is
ubiquitous in nature and mainly prevalent in soil
and human intestine
It best grows in blood agar at human body
temperature
The spores are able to tolerate high heat and
resistance to normal cleaning (except diluted
bleach)
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Transmission
The transmission of C. difficile is mainly due to nosocomially (hospital acquired
infection) at hospitals, nursing homes and other medical institution.
The rate of C. difficile acquisition is estimated to be 13% in patients with hospital stays
up to 2 weeks and 50 % in those with hospital stays longer than 4 weeks
Clostridium tetani
1884 - Arthur Nicolaier isolated the strychnine like toxins of tetanus from free living
anaerobic soil bacteria
1890 - Antonie Carl and Giorgio Rahtone demonstrated the transmission of tetanus
for the first time
1897 - Edmond Nocard showed tetanus antitoxin induced passive
immunity in humans and use of prophylaxis treatment.
1924 - Pdescombey developed the first tetanus toxoid vaccine to prevent the
induced tetanus
Rod shaped
anaerobic but able to
tolerate little amount
of oxygen
Usually gram
positive
Motile by flagella
Heat resistant spore
survive up to
40+years
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Transmission
Pathophysiology
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Clostridium tetani
Transmission:
Contact with non intact skin, usually injuries from
contaminated objects
It is also seen in the umbilical stump of infants in
underdeveloped countries
Incubation time:
It may take from three to 21 days to develop
any symptoms.
In infants, symptoms may take from three days to
two weeks to develop
Symptoms:
The following are the most common symptoms
of tetanus
Stiffness of the jaw
Stiffness of the abdominal and back muscles
Contraction of the facial muscles
Fast pulse
Fever
Sweating
Painful muscle spasms near the wound area
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C. tetani infection/intoxication
Spores of
C. tetani
in wound
Germination and toxin
production under
anaerobic conditions
Transport of toxins via
lymphatic- bloodstream
through motor neurons
to spinal cord
Wound
contaminated
with soil
Symptoms
[Madigan et al. 2009]
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Mechanism of action
Inhibitory interneurons
Excitation signals
from the
central nervous
system
Release of acetylcholine
inhibited by glycine
 Muscle relaxation
Tetanus
toxin
Uncontrolled release
of acetylcholine
 Spastic paralysis
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Modified according to [Madigan et al. 2009]
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Mechanism of action
 Inhibitory neurotransmitter glycine usually stops the
release of acetylcholine from the motor neurons
 muscle relaxation
 AB toxin (= Tetanospasmin, 50 kDa light chain + 100 kDa
heavy chain) inhibits release of glycine from inhibitory
interneurons
 No glycine  constant release of acetylcholine
 spastic paralysis / uncontrolled contraction of the muscle
[Madigan et al. 2009]
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Treatment:
 Immediate treatment with human tetanus immune
globulin (TIG)
 Medication to control muscle spasm
 Aggressive wound care
 Antibiotics: Metronidazole, Clindamycin, Erythromycin, Penicilin
Prevention:
 Being fully immunized is the best tool to prevent tetanus
 Immediate and proper wound care can also help
prevent infection
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Clostridium perfringens
Transmission:
 C. perfringens is one of the most common causes of
foodborne illness
 eating food contaminated with large numbers of
C. perfringens bacteria that produce enough toxin in
the intestines to cause illness
 Beef, poultry, gravies, and dried or pre-cooked foods
are common sources of C. perfringens infections
 C. perfringens infection often occurs when foods are
prepared in large quantities and kept warm for a
long time before serving
Incubation Time:
Persons infected with C. perfringens develop
diarrhea and abdominal cramps within 6 to 24
hours (typically 8-12)
Symptoms:
Diarrhea, usually without vomiting or fever
Abdominal cramps
Nausea
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Mechanism of action
Treatment:
Oral rehydration
in severe cases, intravenous fluids and electrolyte
replacement can be used to prevent or treat dehydration
Prevention:
foods commonly associated with C. perfringens
infections should be cooked thoroughly in temperatures
Sufficient cooling of pasteurized and cooked food
(especially meat products)
No long storage periods
Good cleaning of food
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Clostridium botulinum
Transmission:
Botulism is caused by exposure to botulinum toxin,
humans can become infected in a number of ways:
 Inhalation of toxin
 Consumption of toxin (foodborne)
 Consumption of C. botulinum spores
(infant; adult intestinal toxemia)
 Contamination of a tissue with C. botulinum
spores (wound)
Incubation Period:
 12-80 hours (range 2 hours to 8 days)
Botulism
Foodborne botulism
C. botulinum in food
Toxin produced
Toxin ingested
Toxin in bloodstream
Attacks neurons
(flaccid paralysis)
Infant botulism
C. botulinum spores
ingested
Wound botulism
C. botulinum spores
in wound
C. botulinum grows
in intestinal tract
C. botulinum grows
in wound
Toxin produced
Toxin produced
Modified according to [Salyers et al. 1994]
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Botulinum toxin
7 related AB toxins (A‐G) most potent biological toxins known [Madigan et al. 2009]
Relevant toxins for human diseases: A, B, E [Lindstöm et al. 2006]
Light chain
(50 kD)
A
Heavy chain
(100 kD)
B
AB toxin (150 kD)
[Zhang et al. 2012]
Mechanism of C. botulinum intoxication
 Absorption of toxin by mucosal surfaces in the
gastrointestinal system, the eye or non intact skin.
 Botulinum toxin blocks acetylcholine release at the
neuromuscular junction of skeletal muscle neurons
and peripheral muscarinic cholinergic autonomic
synapses
 It binds irreversibly to presynaptic receptors to
inhibit the release of acetylcholine and cause
neuromuscular weakness and autonomic
dysfunction
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http://microbewiki.kenyon.edu/images/0/0d/BOTULINUM_TOXIN_A_Mechanism_of_Action.jpg
Symtomps:
Nausea
Constipation
Paralysis
Double vision , blurred vision
Dry mucous membranes
Dysphagia
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Treatment:
Foodborne botulism
• Within 1 hour of ingestion of suspected food, the
recommended course of action is a gastric lavage, or
enemas and the administration of a cathartic (sorbitol)
• Administration of antitoxin: only useful, if toxin has not
yet bound to neural cell
Wound botulism
• Antitoxin, wound debridement, and antibiotic treatmen
Infant botulism
• Requires meticulous supportive care
• An investigational human-derived botulinum
immunoglobulin (BIG) is available for the treatment
Prevention:
Foodborne botulism can be controlled by safe
canning and food manufacturing processes
The risk factors for infant botulism are poorly
described. Honey should not be fed to infants
because it has been identified as a food source
Wound botulism cases have occurred in persons
who used illicit drugs
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Clostridium difficile
Transmission:
 C. difficile bacteria and their spores are found in feces.
People can get infected if they touch surfaces
contaminated with feces, and then touch their mouth
 Healthcare workers can spread the bacteria to their
patients if their hands are contaminated
Incubation period:
 The exact incubation time for CDI is unknown,
the time from acquisition to disease is relatively short,
perhaps no longer than 7 days
Symptoms:
Mild C. difficile colitis:
 a low-grade fever
 mild diarrhea (5-10 watery stools a day)
 mild abdominal cramps and tenderness
Severe C. difficile infection:
 Watery diarrhea, up to 15 times each day
 High fever (temperature of 38°C to 40°C)
 Severe abdominal pain
 Loss of appetite
 Blood or pus in the stool
 Weight loss
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Mechanism of infections
Mechanism of action
Toxin A: Enterotoxin that disturbs the transport of
electrolytes  loss of fluid, diarrhea
Toxin B: Cytotoxin that damages cells of the colon
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[Jones et al. 2012]
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Treatment:
 Antibiotics: Metronidazole, Vancomycin
 Probiotics
 Rehydration
Prevention:
 To prevent the spread of C. difficile hospitals
and other health care facilities follow strict
infection control guideline
• Hand washing
• Contact precautions
• Thorough cleaning
DIAGNOSTIC METHODS
ASHIK
TIMILSINA
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CONTENTS
Culture Methods
Test for Clostridia Identification
Gram Staining
Equipment for Anaerobic culturing
Diagnostic Methods
– Mouse Lethality Assay
– Endopeptidase Assay
– Immunological Methods
– Molecular Detection
Culture Media
Clostridia are obligate anaerobes
Media used for isolation are
Non-selective media: AEA Sporulation Broth (Base),
modified, Reinforced Clostridial Agar………
Selective Media: Clostridium difficile Agar (Base),
Anaerobic Blood Agar with Neomycin……..
Differential Media: Egg-Yolk Agar
Selective Differential Media: HiCrome™ M-CP
Agar Base, SPS Agar modified…..
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Egg Yolk Agar
Special kind of culture media
 Contains suspension of egg yolk for identification
of lecithinase and lipase activity of different
clostridium species
 Lecithinase degradation in the yolk results
in opaque colonies
 Lipase enzyme hydrolizes the fat and results
in iridescent sheen of the colony
LEC/LIP Activity of different clostridia species
Species
LEC
LIP
C. botulinum
+
+
C. prefringens +
_
C. tetani
_
_
C. difficile
_
_
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Egg Yolk Agar
Lecithinase activity
Lipase activity
Anaerobic Equipment
•
•
•
•
Anaerobe Atmosphere Generation Bags
Anaerobe Indicator Test
Anaerobe Jar Insert for Petri Disks
Anaerobic Jar
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Gram Staining
C. botulinum
C. difficile
C.tetani
C. perfringens
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Mouse lethality assay (C. botulinum)
• standard procedure to detect C. botulinum
• standard procedure to detect botulinum neurotoxins
• Mice are injected with the cultivated microorganisms
(fluid extract) or with fluid extract of sample
(fecal, serum, gastric, wound, food samples)
• The mice get different antitoxins.
Fluid extract of
cultivated
microorganisms
or sample fluid
Antitoxin
Observed for
48h
Mouse
with
Antitoxin
A
Mouse
with
Antitoxin
B
Mouse
with
Antitoxin
E
Mouse
with
Antitoxin
F
Mouse
With No
Antitoxin
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After 48 h:
Signs of botulism:
 Fuzzy hair
 Muscle weakness
 Respiratory failure ‐> wasp‐like narrow waist
Mouse
with
Antitoxin
A
Mouse
with
Antitoxin
B
Mouse
with
Antitoxin
E
Mouse
with
Antitoxin
F
Mouse
With NO
Antitoxin
Comments
• Generally group I and II clostridium causes
Botulism in humans
• Group III is involved with animal botulism
• Group I culture produces A, B or F toxin
• Group II culture produces B, E or F toxin
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Pro’s and Con’s
Pro’s : Very Sensitive
Con’s: Laborious, expensive, ethical problem,
detects only C. botulinum
Endopeptidase Assay (C. botulinum)
• These assay method is applied for detection of
type A and type B botulinum neurotoxins
• Type B neurotoxin is based on cleavage of
synthetic peptide substrate
• Type A neurotoxin is based on cleavage of peptide
substrate derived from protein SNAP-25
• These peptide cleave shows the type of neurotoxin
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Pro’s and Con’s
Pro’s: Highly specific, no cross reactivity between
different botulinum toxins
Con’s: Needs more research, only
C. botulinum detectable, only biologically
active neurotoxin
Enzyme Linked Immunosorbent Assay (ELISA)
• Combines specificity of antibodies with sensitivity
of simple enzyme essay
• ELISA measures antigen or antibody concentration
• Are used to detect antigens that are recognized
by antibody or vice-versa.
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Pro’s and Con’s
Pro’s: Technical simple, fast to perform and
interpret, C. botulinum, C. perfringens,
C.tetani, C. difficile can be detected by
this technique
Con’s: Inactivated toxins  false positive result
Genetic variation in serotypes  false
negative results
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Molecular Detection
DNA-based detection methods have overtaken all
the conventional techniques
Technique is based on the detection of the clostridia
toxin genes in the sample
Detects clostrida toxin genes:
 botA , botB , botE , botF (C. botulinum)
 tcdA, tcdB (C. difficile)
 cpe, plc, cpa, cpb (C. perfringens)
 tetR (C. tetani)
Sample
DNA Exraction
Polymera
se Chain
Reaction
PCR
Multiplex
PCR
qPCR
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Principle Polymerase Chain Reaction
Primers of PCR for bolinum toxin genes
Toxin type
Primer
Product size (bp)
A
IOAf/IOAr
101
B
CBMLBf/CBMLBr
205
E
CBMLEf/CBMLEFr
389
F
CBMLFf/CBMLFr
543
IAC
IACf/IACr
698
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Multiplex PCR
Multiplex PCR
• Multiple primer pairs are used  more than
one gene can be amplified at the same time
• Specific amplified gene = defined amplicon size
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Quantitative real-time PCR
• One method used is Taq-Man real-time PCR
• Taq-Man probe is used in addition to primers in PCR
• Probe is an oligonucleotide consisting of a
sequence matching to the middle of the template
sequence (DNA)
• Probe has attached: Flurophore and Quencher
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Pro’s and Con’s
Pro’s: Clinic sample can be used directly,
fast procedure, sensitive
Con’s: Expensive (rt‐qPCR), detects only the toxin
gene and not the toxin itself
ASSAY
Time to perfrom
Application
Pro’s
Con’s
Mouse Lethality
1‐4 days
Bacterial culture
serum, gastric
content, food and
environment
sample etc..
Very Sensitive
Laborious, ethical
problem,
expensive, detects
only C.
botulinum
Endopeptidase
1‐2 days
Medical industry,
infant faecal
sample, human
serum etc…
High Specific, no
cross reaction
between
botulinum toxin
Only
C. botulinum
detectable
ELISA
8 hrs
Bacterial culture,
innoculate meat,
cheese food etc..
Technically
simple, fast to
perform and
interpret,
different toxins
detectable
Inactivated toxins
false result
PCR
Fast, few hours
Medical field,
Clinic sample can
laboratory analysis be used directly,
fast procedure,
sensitive, different
clostridia genes
detectable
Expensive
(rt‐qPCR), detects
only the gene, not
the toxin itself
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References
• Clinical Microbiology Reviews – Laboratory
Diagnostics of Botulism [Lindström,Korkeala]
• Manual of Clinical Microbiology
[Patrick R. Murray, Ellen Jo Barron et al.]
• Medizinische Mikrobiologie
[Herbert Hof, Rüdiger Dörries]
• Dario et al, Multiplex PCR for Detection of
Botulinum Neurotoxin – Producing Clostridia in
clinical, food, and environment samples;
(20):6457-6461; Appl. Environmental
Microbiology, 2009
References
•
•
•
•
•
•
Lindström M, Korkeala H: Laboratory Diagnostics of Botulism.
Clin. Microbiol. Rev. 2006, 19(2):298
Madigan MT, Martinko JM, Dunlap P V, Clark DP: Biology of
Microorganisms. 12th edition, Pearson Education Inc.,
San Francisco 2009
Salyers AA, Whitt DD: Bacterial Pathogenesis: A molecular
approach. ASM Press, Washington, D.C. 1994
Zhang Y, Lou J, Jenko KL, Marks JD, Varnum SM:
Simultaneous and sensitive detection of six serotypes of botulinum
neurotoxin using enzyme‐linked immunosorbent assay‐based
protein antibody microarrays. Analytical Biochemistry 430 (2012) 185–192
“Development of real‐time PCR tests for detecting
botulinum neurotoxins A,B,E,F producing Clostridium botulinum,
[…].” [P.Fach et al.], Journal of AppliedMicrobiology
Taq‐Man‐Picture [http://en.wikipedia.org/wiki/File:Taqman.png]
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