Clostridium tetani

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Clostridium tetani:

Clostridium tetani Dr Sonny

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Clostridium tetani Kingdom: Bacteria Phylum: Firmicutes Class: Clostridia Order: Clostridiales Family: Clostridiaceae Genus: Clostridium Species: C. tetani ~thick peptidoglycan cellular wall

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Clostridium tetani

History :

History Clostridium tetani Tetanus was known since ancient times 1884- Nicolair suggested that disease was due to strychnine like toxin produced locally Isolated toxin from anaerobic free living soil bacteria 1886- Rosenbach first demonstrated the slender bacillus with round terminal spores

History :

History Clostridium tetani 1889- Kitasato isolated the organism in pure cultures 1890- Von behring and Kitasato described active immunisation

History :

History Clostridium tetani

History :

History Clostridium tetani In 1897, Edmond Nocard showed that tetanus antitoxin induced passive immunity in humans

History :

History Clostridium tetani Passive immunisation was used for treatment & prophylaxis during world war 1

History :

History Clostridium tetani Tetanus toxoid vaccine was developed by P. Descombey in 1924 Widely used during world war 2

Physiology :

Physiology Clostridium tetani Strict anaerobes Do not posess cytochrome oxidase Energy is obtained by ion transport mechanisms & by fermentation of organic compounds Lack catalase

Habitat :

Habitat Clostridium tetani Widely distributed in soil Intestines of human beings and animals Street dust, hospital dust, cotton , wool , bandages , catgut Harmless contaminant of wounds

Morphology :

Morphology Clostridium tetani Gram positive Slender bacillus with 4-8 x0.5um size Non capsulated Motile by peritrichate flagella {except type 6} Gram variable, pleomorphic Spore forming

Morphology :

Morphology Clostridium tetani

Morphology :

Morphology Clostridium tetani Spores are spherical , terminal and bulging Drum stick appearance

Morphology :

Morphology Clostridium tetani

Cultural characters :

Cultural characters Clostridium tetani Obligate anaerobe Optimum temperature 37 0 c PH 7.4 Growth is enhanced by blood & serum Grows well in liquid media where 0 2 have been removed by boiling and autoclaving Liquid media with reducing agents Cysteine , Thioglycollate , Metallic iron, Disodium sulphide , Sodium ascorbate ,

Cultural characters:

Cultural characters Clostridium tetani Growth on solid medium Growth is slow & has a tendency to swarm over Thin effuse spreading film of growth with delicately filamentous advancing edge

Cultural characters:

Cultural characters Clostridium tetani Nutrient agar Granular or ground glass appearance Blood agar 4-6 mm diameter, flat, translucent gray with matt surface, narrow zone of beta hemolysis Irregular rhizoid margins

Cultural characters:

Cultural characters Clostridium tetani

Cultural characters:

Cultural characters Clostridium tetani Nutrient agar Granular or ground glass appearance Egg yolk agar No opalescence or pearly layers

Cultural characters:

Cultural characters Clostridium tetani Growth in RCM Turbidity Some gas formation Meat is turned black on prolonged incubation Meat is reduced in volume Foul smelling end products

Cultural characters:

Cultural characters Clostridium tetani Saccharolytic reaction It causes fermentation of glycogen of muscles Production of acid and gas Meat particles remain intact e.g Cl. perfringens Proteolytic Reaction It causes digestion of meat particles Formation of black colour , foul smelling due to sulfur compounds

Cultural characters:

Cultural characters Clostridium tetani Deep agar shake cultures Colonies are spherical fluffy balls, 1-3mm diameter having filaments with radial arrangement Gelatin stab culture Fir tree type of growth with slow liquefaction

Cultural characters:

Cultural characters Clostridium tetani Fildes technique Water of condensation at the bottom of NA slant is inoculated with mixed cultures and incubated anaerobically Sub culture from the top of the tube will yield pure growth of tetanus

Cultural characters:

Cultural characters Clostridium tetani Mixed cell cultures are inoculated laterally on BA Tetanus bacilli spread over the entire plate as a thin film with dentate spreading edge

Cultural characters:

Cultural characters Clostridium tetani Inhibition of swarming Increasing the concentration of agar Addition of chemicals Addition of agglutinating antibody to the medium Drying the surface of the medium Alcohol plates Chloroform vapour exposure to exclude Pseudomonas & Proteus

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Clostridium tetani Classification 10 serological types have been described Based on flagellar antigen Type 6 is non motile

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Clostridium tetani Biochemical reactions Most tests are negative Sugars are not fermented cooked meat is digested Lecithinase , lipase, nitrate –negative Gelatin is liquefied slowly Forms indole MR & VP negative

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Clostridium tetani

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Clostridium tetani End products of peptone yeast extract broth culture- Acetic acid Propionic acid Butyric acid Helps in identification by gas liquid chromatography

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Clostridium tetani Resistance Most spores are destroyed by boiling for 10- 15 mnts Some resist boiling for 3 hrs Autoclaving at 121 o c for 20 mnts ensures spore destruction

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Clostridium tetani Sporicidal agents Iodine (1% aqueous solution) Hydrogen peroxide (10 volumes) Formaldehyde Gluteraldehyde Peracetic acid

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Clostridium tetani Toxins Tetanolysin Tetanospasmin Non spasmogenic peripherally active neurotoxin

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Clostridium tetani Tetanolysin Heat labile Oxygen labile Antigenically similar to hemolysins of Cl.perfringens , Cl.novyi , & Strep pyogenes Not relevant in pathogenesis

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Clostridium tetani Tetanospasmin Responsible for tetanus Oxygen stable Heat labile Zinc metalloprotease

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Clostridium tetani Gene is located on 75 kb plasmid Initially single polypeptide Later autolysed to heavy chain & light chain joined by disulphide bond

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Clostridium tetani

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Clostridium tetani Binds to presynaptic membrane of motor neuron Retrograde axonal transport to cell bodies Toxin diffuses to the terminals of inhibitory cells Degrades synaptobrevin Release of inhibitory neuro transmitters are blocked

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Clostridium tetani Diminished inhibition -resting fire rate of motor neurons increases -rigidity Abolition of spinal inhibition leads to uncontrolled spread of impulses initiated any where in CNS spasms due to simultaneous contraction of agonists & antagonists MLD for humans 130 nanograms

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Clostridium tetani

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Clostridium tetani

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Clostridium tetani

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Clostridium tetani

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Clostridium tetani Pathogenesis No invasive property Spore germination & toxin production Occurs in Reduced oxidation reduction potential Devitalized tissues Foreign bodies Concurrent infections

Clinical features :

Clinical features Clostridium tetani Tetanus IP-6-12 days can be as long as 30 days Predisposing factors Puncture wounds Unsterile injections Application of cow dung on umbilical stump Lapse in asepsis following surgeries, ear boring , Circumcision, Otitis media, septic abortion Complicates skin ulcers, abscesses, gangrene Drug abuse (skin popping)

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Clostridium tetani Clinical classification Localised Generalised Cephalic Neonatal

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Clostridium tetani Generalised Begins with risus sardonicus & trismus Generalised spasms Ophisthotonic posturing with flexion of the arm & extension of the legs Patient remains conscious Upper air way obstruction Diaphragm participate in spasms

Sir Charles Bell's portrait of a soldier dying of tetanus. The characteristic rigidity of the body is referred to as opisthotonos and risus sardonicus. Original in the Royal College of Surgeons of Edinburgh, Scotland.:

Sir Charles Bell's portrait of a soldier dying of tetanus. The characteristic rigidity of the body is referred to as opisthotonos and risus sardonicus . Original in the Royal College of Surgeons of Edinburgh, Scotland . Clostridium tetani

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Clostridium tetani

Neonatal tetanus:

Neonatal tetanus Clostridium tetani a form of generalized tetanus that occurs in newborns. Infants who have not acquired passive immunity because the mother has never been immunized are at risk. usually occurs through infection of the unhealed umbilical stump when the stump is cut with a non-sterile instrument. common in many developing countries and is responsible for about 14% (215,000) of all neonatal deaths [7]

Local tetanus:

Local tetanus Clostridium tetani an uncommon form of the disease have persistent contraction of muscles in the same anatomic area as the injury generally milder; only about 1% of cases are fatal may precede the onset of generalized tetanus.

A preschool-aged boy with localized tetanus secondary to the parent attempting to drain an impetigo lesion with a mesquite thorn contaminated with tetanus spores. :

A preschool-aged boy with localized tetanus secondary to the parent attempting to drain an impetigo lesion with a mesquite thorn contaminated with tetanus spores. Clostridium tetani

Cephalic tetanus:

Cephalic tetanus Clostridium tetani rare form of the disease otitis media , injuries to the head. involvement of the cranial nerves, especially in the facial area.

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Clostridium tetani Lab diagnosis Made on clinical grounds Lab tests help in confirmation Microscopy Culture Animal inoculation

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Clostridium tetani Elevated leukocyte count CSF study normal Electromyogram Continuous discharge of motor units Absence of silent interval after an action potential elevated muscle enzymes

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Clostridium tetani Serum anti toxin level Anti toxin level >0.15U per ml -protective

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Clostridium tetani Differential diagnosis Alveolar abscess Strychnine poisoning Dystonic drug reactions Meningitis Encephalitis Rabies

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Clostridium tetani Markedly increased tone of central muscles with relative sparing of hands & feet strongly suggests tetanus

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Clostridium tetani Microscopy Unreliable Demonstration of drumstick bacilli Morphologically similar bacilli- Cl.tetanomorphum , Cl.sphenoides

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Clostridium tetani Immunofluorescence staining Batty & walker 1965 Commercially available immmunoglobulins Fix the smear in acetone for 10 mnts Spread a drop of conjugated immunoglobulin - leave for 30mnts

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Clostridium tetani Wash with buffered saline Blot dry Mount with buffered glycerol saline & plastic cover slip

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Clostridium tetani Culture Specimen: tissue bits from necrotic depths of wounds Aspirates in syringes Media to be inoculated BA 2 selective BA CMB Antitoxin controlled plate

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Clostridium tetani Inoculation on blood agar Material is inoculated on one half of BA Swarming growth is seen on opposite half after 1-2 days of incubation anaerobically

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Clostridium tetani One selective BA plate is left undisturbed for 48 hrs Check the other plates after 24 hrs of incubation & daily for up to 4 days Make a pure sub culture from the spreading edge to pre reduced BA & CMB for 48 hrs

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Clostridium tetani Examine CMB daily by microscopy If clostridial forms are seen 1 Direct immmunofluorescence staining 2 Heat part of the culture at 80 o c for 10 mnts Sub culture heated & unheated samples on BA 3 Toxin testing Can be done directly from supernatant of CMB enrichment culture

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Clostridium tetani Or wait until CMB is pure Set up 1 toxin tests 2 biochemical tests 3 fatty acid profile by GC

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Clostridium tetani Inoculation on RCM 3 tubes are used First-80 0 c for 15mnts Second-80 0 c for 5 mnts Third – left unheated Subculture on one half of BA pates daily for up to 4 days Subculture from the swarming edge of the growth

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Clostridium tetani Selective media Incorporation of polymyxin B

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Clostridium tetani Identification & toxigenicity testing BA with 4% agar having tetanus antitoxin spread over one half Strains are stab inoculated to each half of plate Hemolysis around the colonies on the half without antitoxin Indicates the production of tetanolysin unreliable

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Clostridium tetani Toxigenicity testing in animals Guinea pigs Or mice 0.2ml of 5- 10 day old cooked meat broth culture To the right of the base of tail of mice Stiffness of the tail & hind limbs paralysis of tail & hind limb Spine of the animal tend to curve towards right

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Clostridium tetani Control animal Protected with 500 - 1500 units 0f anti toxin 1 hour before the test Intracutaneously /intra peritoneally

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Clostridium tetani Treatment Goals Eliminate source of toxin Neutralize unbound toxin Prevent muscle spasms Protection of airway Wound exploration & debridement

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Clostridium tetani Antibiotic therapy Pencillin 10- 12 million units-10 days Metronidazole 500mg every 6 hrs Clindamycin , erythromycin Additional antibiotics for concurrent infection with other organisms

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Clostridium tetani Antitoxin Neutralise unbound toxin Lowers mortality Dose: 10000 units IM Administer before manipulating the wound Half life of anti toxin is long No additional doses are required

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Clostridium tetani On recovery- active immunization Immunity is not induced by small amount of toxin that produce the disease

Tetanus Toxoid:

Tetanus Toxoid Clostridium tetani • Formalin-inactivated tetanus toxin • Schedule Three or four doses + • Booster Booster every 10 years • Efficacy Approximately 100% • Duration Approximately 10 years • Should be administered with diphtheria toxoid as DTaP , DTP or Td

DTaP, DT, and Td:

DTaP , DT, and Td Clostridium tetani Diphtheria Tetanus DTaP , DTP 7-8 Lf units 5 Lf units Td (adult) 2 Lf units 5 Lf units ƒ Pertussis vaccine and pediatric DT used upto age 6 ƒ Adult Td used for persons 7 years and older.

Routine DTaP Primary:

Routine DTaP Primary Clostridium tetani Vaccination Schedule Dose Age Interval Primary 1 6wks -- Primary 2 10wks 4wks Primary 3 14wks 4 wks Primary 4 15-18 months 6mnths

Routine DTaP Schedule:

Routine DTaP Schedule Clostridium tetani Children <7 years of age Booster Doses 4-6 years, before entering school 11-12 years of age if 5 years since last dose (Td) Every 10 years thereafter (Td)

Children Who Receive DT:

Children Who Receive DT Clostridium tetani •The number of doses of DT needed to complete the series depends on the child’s age at the first dose: • if first dose given at <12 months of age, 4 doses are recommended • if first dose given at >12 months, 3 doses complete the primary series

Routine Td Schedule:

Routine Td Schedule Clostridium tetani Unvaccinated Persons >7 Years of Age Dose Interval ------- ---------- Primary 1 --- Primary 2 4 wks Primary 3 6-12 mos Booster dose every 10 years

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Clostridium tetani Passive immunization Treatment dose: 10000 U Prophylactic dose: 250 U Protective antibody levels lasts for 4- 6 weeks Vaccine & TIG should be administered at separate sites with separate syringes

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Clostridium tetani Wound management Depends on immune status A patient is immune for 6 months after first two injections of toxoid 5- 10 years after 3 injections of toxoid 2-3 weeks after equine anti toxin 6- 8 weeks after human antitoxin

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Clostridium tetani

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Clostridium tetani Prophylaxis Surgical attention Antibiotics Immunisation

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Clostridium tetani Maternal and Neonatal Tetanus Elimination by 2005

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Clostridium tetani Neonatal tetanus elimination: The reduction of neonatal tetanus cases to fewer than 1 case per 1,000 live births in every district of every country.

Neonatal tetanus:

Neonatal tetanus Clostridium tetani Tetanus occurring in a newborn between the 3rd and 28th day after birth

Maternal tetanus:

Maternal tetanus Clostridium tetani Tetanus that strikes women during pregnancy or within six weeks of the termination of pregnancy.

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Clostridium tetani Maternal tetanus is responsible for at least 5 per cent of maternal deaths, approximately 30,000 deaths annually

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Clostridium tetani In 1988 -787,000 newborns died of neonatal tetanus estimated annual global NT mortality rate was approximately 6.7 NT deaths per 1000 live births - c 1989-the 42nd World Health Assembly called for elimination of neonatal tetanus by 1995

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Clostridium tetani the target date for MNT elimination was postponed to 2000 In 2000 the Initiative was re-constituted and elimination of maternal tetanus was added to the goal with a 2005 target date

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Clostridium tetani

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Clostridium tetani 2008 59,000 newborns died from NT a 92% reduction from the situation in the late 1980s 46 countries still had not eliminated MNT in all districts by December 2010, 39 countries have not reached MNT elimination status

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Clostridium tetani strategies for achieving MNT elimination include: Strengthening routine immunization of pregnant women with tetanus toxoid vaccine (TT) TT Supplementary Immunization Activities (SIAs) in selected high risk areas, targeting women of child bearing age with 3 properly-spaced doses of tetanus toxoid Promotion of clean deliveries Reliable NT surveillance

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Clostridium tetani Territories in India validated as having eliminated MNT: Andhra Pradesh , Chandigarh & Goa , Gujarat , Haryana , Himachal Pradesh , Karnataka , Kerala , Lakshadweep , Maharashtra , Pondicherry, Punjab & Sikkim , Tamil Nadu & West Bengal ,

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Clostridium tetani

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Clostridium tetani Thanks

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Clostridium tetani

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Clostridium tetani

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