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Clostridium Clostridium perfringensperfringensClostridium Clostridium botulinumbotulinum
Dr Kamran Afzal
Classified Microbiologist
Clinically important Gram Clinically important Gram positive bacillipositive bacilli
Spore forming1. Bacillus2. Clostridium
Non spore forming1. Corynebacterium2. Listeria
Bacilli w/ branching filaments
1.Actinomyces
2.Nocardia
2
SporesSpores
SporesSpores
Clostridia form endospores under adverse
environmental conditions
Spores are a survival mechanism
Spores are characterized on the basis of
position, size and shape
Most Clostridium spp., including Cl.
perfringens and Cl. botulinum, have ovoid
subterminal (OST) spores
Cl. tetani have round terminal (RT) spores
Classification Based On The Classification Based On The Type Of Disease ProducedType Of Disease Produced
A . Tetanus Cl. tetani - Present in soil
B. Gas gangrene◦ Established Cl. perfringens ‘gut’ organism
Cl. septicumCl. novyi
- Less pathogenic Cl. histolyticumCl. fallax
- Doubtful Cl. bifermentansCl. sporogenes
C. Food poisoning 1. Gastroenteritis Cl perfringens Type
A
2. Botulism Cl botulinum Soil
3. Pig-bel Cl perfringens Type
C
D. Acute colitis - Cl difficile Gut (pseudomembranous colitis)
◦ Commonest cause of ‘nosocomial’ diarrhoea
Introductory CharacteristicsIntroductory Characteristics
Obligate anaerobes Gram positiveCapable of
producing endospores
Rod-shaped◦ Greek word for
spindle, kloster
Club-shaped◦ Endospores form club
end
Clostridium Associated Human Clostridium Associated Human DiseasesDiseases
Clostridium perfringensClostridium perfringens
CharacteristicsCharacteristics
Gram positiveRod-shapedNon-motileAnaerobicCapsulatedDouble zone of haemolysisFive types of strains
A - EFour lethal toxins
Alpha, Beta, Epsilon and Iota
EpidemiologyEpidemiology
Lives in soils esp manured and cultivated lands
Persists in human and animal intestinal tracts and faecal matter
Can survive higher temp (450C)
Doubling time can be as short as 8 minutes
Food poisoning strains produce spores that can resist boiling for several hours
Gas gangrene strains produce spores that are inactivated by boiling for few minutes
Virulence FactorsVirulence FactorsM
ajo
rM
ino
r
• Enzymes•Hyaluronidase, Proteinase and Collagenase
Lab DiagnosisLab Diagnosis
•Large rectangular gram-positive bacilli
Inner beta-hemolysis = θ toxin Outer alpha-hemolysis
= α toxin
•Double zone of haemolysis
• Lack of inflammatory cells
•Nagler Reaction (egg-yolk agar)
NOTE: Lecithinase (α-toxin; phospholipase) hydrolyzes phospholipids in egg-yolk agar around streak on right. Antibody against α-toxin inhibits activity around left streak
Cl perfringensCl histolyticumCl septicumCl novyii
Clostridial food poisoningClostridial food poisoning
Cl perfringensHeat resistant Enterotoxin Carriers for food poisoning strainsSurvival of heat resistant spores in bulk
mealsSporulation in gut - Short IP and watery
diarrhoea for 24-48 hoursUsually symptoms occur within 6-24 hours of
ingestion and can last ~24 hours
Salmonella sppStaphylococcus aureusCampylobacter sppClostridium perfringens
Necrotizing enteritis (Pig-bel)Necrotizing enteritis (Pig-bel)
New Guinea natives have pork feasts
Clostridia survive in under-cooked meat
Production of Beta toxin by Cl perfringens
Type C
Beta toxin acts on small intestine
Gas GangreneGas Gangrene
Gas gangrene C. perfringens type A (Principal),
Capsulated, non-motile
Lecithinase C - toxaemia
Nagler reaction
Colonies with haloes
Colonies withouthaloes
Incorporated withAntitoxins
PathogenesisPathogenesis
Dead tissue, blood clots, aerobic organisms
In an injury
Development of ANAEROBIC CONDITION
(Exogenous infection) Germination of spores
Gas gangrene
oedema, necrosis, gas production
toxaemia, myositis
crepitus
ManagementManagement Prompt surgical intervention
◦ Sutures are removed, fascial compartments are incised to release tension, necrotic tissue is debrided
Hyperbaric oxygen◦ Special pressurized chamber
Antitoxin antiserum◦ A polyvalent antitoxin antiserum containing Cl
perfringens, Cl novyii and Cl septicum antitoxin
◦ Now replaced with intensive antimicrobial therapy
Antimicrobial therapy
Antibiotics to cover Clostridia and other contaminants
PenicillinMetronidazoleAminoglycoside
OrClindamycin
Or broad spectrum lactum cefotaxime, imipenem
Used to give antitoxins to cover C. perfringens, C. septicumand C. novyi
Clostridium botulinumClostridium botulinum
EpidemiologyEpidemiology
Found in soil, sediments of lakes, ponds,
coastal waters, decaying vegetation
Intestinal tracts of birds, mammals and
fish
Usually seen in canned foods
◦ Hams, sausages, fish, liver paste, honey and
home preserved meat products and
vegetables
CharacteristicsCharacteristics
Anaerobic Gram-positive bacillus that forms oval sub-terminal spores
Botulinum Toxin - One of the most
poisonous natural substances known
Seven toxigenic subtypes of the organism◦ A, B, C, D, E, F and G
◦ The principle cause of human disease A,B and E
SporesSpores
Spores can withstand boiling in water for several hours
Destroyed by moist heat at 120 0C within 5 min
Insufficient heating in the process of preserving foods – Botulism
Canning factories should ensure ‘adequate heating’ in all parts of canned contents
Resistance of some spores to irradiation
Action of ToxinAction of Toxin
Structure: Synthesized as a
polypeptide chain that cleaves
into two chains, a light and
heavy linked by disulfide bonds
Blocks release of Ach
◦ Failure to release neurotransmitter
◦ Zinc-dependent endopeptidase that
cleaves synaptobrevins
Flaccid Paralysis
Permanent binding and
damage
Mechanism of Action of ToxinMechanism of Action of Toxin Toxin is neurotoxic protein Not inactivated by stomach / intestinal enzymes Destroyed by heating at 1000C for 20 mins Action :Block release of Acetylecholine at synapses and
NMJ of peripheral and cranial nerves - flaccid paralysis
SymptomsSymptoms
Begin 8-36 hours after ingestionLength: 2 hours to 14 days after entering
circulationPreliminary symptoms: weakness, dizziness,
dryness of mouth, nausea, vomitingAfter Neurological disturbance: blurred
vision, inability to swallow, difficulty in speech, descending weakness of skeletal muscles
Respiratory paralysis and cardiac failure - death
Clinical SyndromesClinical SyndromesFlaccid paralysis
Food-borne: ingestion of foods in which spores have
germinated and grown in, considered an intoxication –
most common form
Wound: infects a wound and then produces toxins that
spread through the bloodstream – very rare
Infant: infection establishes itself in the bowels of
infants, colonizes and produces the toxin – common
source is honey
Unidentified: source is unknown, usually from
intestinal colonization with in vivo production of toxin –
usually from surgeries
Lab diagnosisLab diagnosis
Clinical samples
◦ Suspected food, faeces or vomitus
◦ Cl botulinum cultured anaerobically
Toxin detection
◦ Toxin-Anti toxin neutralization by inoculation in mice
TreatmentTreatment Priorities
◦ To remove unabsorbed toxin from stomach and small intestine
◦ To neutralize unfixed toxin by giving polyvalent antitoxin (A,B and E)
◦ To give relevant intensive care and support
Penicillin or other beta-lactam antibiotics?
A prophylactic dose of polyvalent antitoxin should be given to all the persons who have eaten ‘suspicious food’ containing botulism toxin
Alternative botulism usesAlternative botulism uses
Botox
◦ A-Type botulism is an
active ingredientBiological Warfare
◦ Poisonous to humans
◦ World War II gelatin capsules with a lethal dose slipped into food or drink 1 gram crystalline toxin dispersed evenly and
inhaled = 1 million deaths
What are the causative agent(s) of gas gangrene?◦Cl. perfringens◦Cl. novyii◦Cl. difficile◦Cl. septicum◦Cl. botulinum
What are the causative agent(s) of gas gangrene?◦Cl. perfringens◦Cl. novyii◦Cl. difficile◦Cl. septicum◦Cl. botulinum
Which one is not a Clostridial toxin?◦Neurotoxin◦Histotoxin◦Aflatoxin◦Enterotoxin
Which one is not a Clostridial toxin?◦Neurotoxin◦Histotoxin◦Aflatoxin◦Enterotoxin
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 (), beta (), epsilon (), and iota () toxins) and an enterotoxin • six minor toxins (delta(), theta(), kappa(), lambda(), mu(), nu()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??)
C. botulinum — agent of botulism, a rare, but severe (lethal) neuroparalytic disease
Morphology and Physiology • heterogeneous group of fastidious, strictly anaerobic bacilli • motile by peritrichous flagella • heat-resistant spores (ovoid, subterminal) • proteolytic and non-proteolytic
Antigenic Structure • species divided into four groups (I-IV) based on type of toxin produced and proteolytic activity • seven antigenically distinct botulinum toxins (types A to G) • somatic antigens - heat stable and heat labile; spore antigens - more specific
Pathogenicity Determinants • lethal foodbome intoxication with toxin types A,B,E,or F; shorter incubation period, poor prognosis • phage-mediated, systemic-acting A-B neurotoxin (botulinum toxin = botulin) released at cell lysis
Mode of Action - one of most extremely potent neurotoxins known (1 ng of purified toxin contains about 200,000 minimal lethal doses (MLDs) for a 20g mouse) • A-B toxin ingested, binds specific receptors on peripheral cholinergic nerve endings
(neuromuscular junctions) where it blocks release of presynaptic acetylcholine (excitatory neurotransmitter) blocking muscle stimulation & resulting in flaccid paralysis
• Early: nausea, vomiting, weakness, lassitude (lack of energy), dizziness, constipation • Later: double vision, difficulty in swallowing and speaking • Final: death due to respiratory paralysis
Lab Identification • microscopic detection or Cx (culture) are often unsuccessful (few organisms and slow growing) • toxin detected and typed in lab via toxicity and antitoxin neutralization tests in mice or by ELISA
Diagnosis/Treatment/Prevention • crucial to rapidly diagnose (symptoms often confusing); note the type of botulinum toxin involved • Tx (treatment) should be administered as quickly as possible on basis of clinical Dx (diagnosis) ventilatory support & trivalent (A, B, E) antitoxin (polyvalent) binds free toxin in bloodstream administer gastric lavage & metronidazole or penicillin eliminates organisms from Gl tract care in home canning and in heating of home-canned food; toxoid is available