AB2 Bacteriology Clostridium

• Clostridium


  Gram-positive, rod-shaped, anaerobic, spore-forming, saprophytic in the environment, commensal in the GI tract, pathogenic for Man and other animals, virulence factors = exotoxins
• Pathogenic clostridia


  • C. perfringens
  • C. novyi
  • C. septicum
  • C. chauvoei
  • C. sordellii
• Enteropathogenic clostridia


  • C. perfringens
  • C. difficile
• Neurotoxic clostridia


  • C. tetani
  • C. botulinum
• Histotoxic clostridia


  • Tissue-invading/wound infections
  • Involve trauma to tissues which allows inoculation or activation of spores
  • Vegetative cells and spores present in the alimentary tract lead to deposition of spores in tissues
  • Spores persist until eliminated (this may take 6 weeks)
  • Tissue injury during the period results in bacterial growth and disease
  • Diseases produced by these bacteria depend on the tissues colonised, the type of trauma and the toxigenicity of the clostridial species
• Lecithinase and necrotising toxins


  Two major groups of toxins found in histotoxic clostridia
• Many of the toxins present in different clostridial species are closely related immunologically
• Protection is not exclusively antitoxic
• C. novyi type B infection (Black's Disease of sheep)

  • Spores germinate in the tracts left by migrating immature fluke (Fasciola hepatica)
  • Toxin production extends the lesion
  • Tissue necrosis
  • Systemic release of toxin
  • No real warning signs; usually fatal
• C. chauvoei (Blackleg)


  • Typically a disease of young (6 months – 2 years) cattle/sheep
  • Often restricted to certain pastures
  • Can occur without obvious traumatic injury- spores considered to be present in the muscle
  • Affected muscles black/dark red and contain gas (crepitant)
• Gangrene


  • C. perfringens, C. novyi, C. chauvoei and C. septicum can cause myositis (muscle inflammation) or gas gangrene
  • Bacteria are introduced directly into the tissues by trauma, e.g. wound
  • All colonise putrefying tissues
  • Pathology of lesions (gas formation, tissue breakdown and proteolysis - SMELL) are often characteristic
  • Serious wound infections in several species (e.g. horses) can be attributed to Clostridium spp.
• Injection of clostridial spores in contaminated heroin: 35 deaths in UK in 2000 (many were in Scotland)
• Control of clostridial infections often involves vaccination with bacterins and toxoids
• Clostridium tetani


  Anaerobic, Gram-positive, straight, slender rod with rounded ends, Spore-forming with terminal 'Drumstick' spores - highly resistant, Peritrichous flagella allowing motility – swarms on blood agar
• Found in intestine of Man and animals, Primary source is animals faeces and soil, especially clay (alkaline). Spores widespread
• Pathogenesis of Clostridium tetani

  1. Spore contaminate wound

  2. Spores germinate in wound – low O2 tension and non-viable material

  3. C. tetani is not an invasive organism – stays at site of initial infection

  4. Toxin production and release

  5. Toxin absorbed from site and moves via peripheral motor neurons within axon to CNS
• Tetanus toxin


  • Two toxins: main pathogenic toxin is tetanospasmin (other is tetanolysin)
  • Tetanospasmin - a Heavy and Light chain
  • Not toxic by oral route – readily degraded
  • Toxin acts by preventing the release of inhibitory neurotransmitters (from inhibitory interneurons) resulting in over-stimulation of motor neurons in CNS
  • Heavy Chain for trafficking and light chain to cleave synaptobrevins
• Tetanus toxin effects


  • Prevents relaxation of muscle fibres - interferes with normal inhibition of motor impulses leading to tonic spasm
  • Affects voluntary muscle. Head and neck usually first areas to be affected (lockjaw)
  • Toxin travels via blood and lymph to all nerves of the body and reaches the CNS via uptake through neuromuscular nerve endings and subsequent intra-axonal route
  • Severity dependent on the amount of toxin reaching the CNS
• Even very minor injuries can introduce spores
• Laboratory diagnosis of Clostridium tetani

  • Gram stained smears – 'drumstick' (presumptive evidence)
  • Direct culture onto blood agar (swarming growth)
• Treatment of tetanus


  Debride, relaxants, (antitoxin) (antibacterials)
• Prevention/control of tetanus


  • Hygiene
  • Toxoid
  • Antitoxin
• Horses are usually protected by two priming doses of tetanus toxoid vaccine, followed by a booster after one year and then every two to three years
• Once tetanus toxin is bound it cannot be neutralised by the anti-toxin. The anti-toxin itself provides protection for about 4 weeks. Ruminants are less susceptible, but tetanus can occur following castration, docking or dystocia in cattle and sheep
• Clostridium perfringens


  • Ubiquitous, in GI tracts of Man and animals, in soil and dust (as spores)
  • Often can 'appear' Gram-negative and rarely forms spores in lab (has G+ wall structure)
  • Lecithinase-producer: forms opalescence on egg yolk agar
• Major toxins of C. perfringens

  • alpha-toxin - lecithinase that acts directly on cell membranes
  • beta-toxin - common to strains causing enteritis, reduces intestinal motility
  • gamma-toxin - acts on endothelium
  • delta-toxin - targets host cell cytoskeleton, alters epithelial permeability
• Many of these diseases have a rapidly fatal course - diagnosis is often carried out by: a) demonstrating specific toxins within the intestinal tract at necropsy, b) detecting toxin in faeces, c) sometimes by isolation of the bacteria and demonstration of toxin type in culture
• Types of Clostridium perfringens based on toxins produced

  • Type A - alpha toxin, food poisoning (Man), enteritis (calf, piglet), gangrene
  • Type B - alpha, beta, epsilon toxins, lamb dysentery, dysentery (foal, calf)
  • Type C - alpha, beta toxins, struck (sheep, goats), dysentery (lamb, calf, pig)
  • Type D - alpha, epsilon toxins, enterotoxaemia, pulpy kidney (sheep, goats)
  • Type E - alpha, iota toxins, doubtful pathogen, but perhaps enteritis (calves, rabbits)
• Enterotoxaemia or pulpy kidney of sheep (C. perfringens Type D)


  • True enterotoxaemia
  • Small numbers of commensal C. perfringens Type D in the intestine
  • Most cases occur in lambs with access to rich pasture
  • Excess digestible carbohydrate entering the small intestine initiates disease; stimulates growth of these saccharolytic bacteria
  • Type D strains reach up to 10^9 per g
  • Elaborate toxins; Epsilon toxin = a protoxin that is activated by intestinal trypsin or chymotrypsin, can accumulate in the brain – neurotoxic effects
  • Local epithelial damage precedes and facilitates absorption of toxin and damage to endothelium, particularly of kidney and brain
  • Hyperglycaemia due to enhanced glycogenolysis leads to glucosuria, and large numbers of non-sporing clostridia in the intestinal tract
  • Animals dying of enterotoxaemia putrefy rapidly
• Dysentery / Enteritis (C. perfringens Types B and C)

  • Produce intestinal lesion due to the beta-toxin
  • Pathogenesis is similar to Type D infections
  • Most cases occur in single lambs of lowland breeds, in suckling piglets or adult sheep
  • The mucosal surface of the small intestine is colonised, beta-toxin characteristically causes mucosal necrosis and ulceration
• C. perfringens Type A "Food Poisoning Strains"

  • Human commensal and most domestic animals occasionally involved in food poisoning (in humans)
  • Often large outbreaks
  • Warm food - contamination by spores
  • Bacteria sporulate in small intestine and produce an enterotoxin
  • Incubation: 8-22h; duration: 1-2 days
  • Severe abdominal cramps with diarrhoea
  • Destruction of villus tips with resultant pain and diarrhoea
• Summary activity of main C. perfringens toxins
• Clostridium difficile


  • Antibiotic-associated diarrhoea pseudo-membranous colitis (PMD) in humans
  • Major issue in hospitals occurs following treatments
  • The organism gains a competitive advantage due to removal of competing microflora
  • Incidence on the decline in the UK due to better management
• Antibiotic‐induced infections, in particular Clostridium difficile as a result of upsetting the balance of microbial population in the gastrointestinal tract
• Clostridium botulinum


  • Anaerobic Gram-positive spore forming bacterium
  • Classified by type of neurotoxin (A-F)
  • Widely distributed in Nature - soils, lake sediments, vegetables, GI tract of mammals, birds and fish
  • Related to other Clostridium spp. Can only be differentiated by toxins
• Animals affected by different Clostridium botulinum toxin types


  • Type A, B, C, D - horses
  • Type C and D - cattle (+A and B)
  • Type C - sheep
  • Type C and D - birds (chickens, wildfowl)
  • Type E - fish
  • Type A, B, E (F) - human
• Botulinum neurotoxins (BoNT)


  • Antigenically different from each other with similar pharmacological effects: A,B, C1,D,E,F
  • Block neurotransmitter release at cholinergic nerve terminals
  • H and L chains - H binds and L is zinc-dependent endopeptidase
  • Proteolysis of specific proteins forming synaptic vesicle docking and fusion complex: e.g. synaptobrevin (B, D & F), SNAP-25 (C1) and syntaxin (A & E)
  • Chromosomally encoded: A, B, E
  • Bacteriophage-encoded: C1 and D
• Wound botulism is rare, but increasing in drug-injecting humans
• Food-borne botulism


  • Ingestion of pre-formed toxin - the most powerful natural toxin – 1g lethal for a human
  • Human disease associated with A, B and E
  • Birds and domestic animals C and D (poorly absorbed by human mucosa)
  • Classic food-borne botulism leads to flaccid paralysis
• Toxico-infectious botulism


  • Wound botulism - 'war' wounds and injecting drug users, toxin produced locally leading to systemic effects
  • Infant botulism
  • Shaker foal syndrome
  • Equine grass sickness - toxin produced in gut leading to systemic effects