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