Spindle shaped CLOSTRIDIUM INTRODUCTION • Genus clostridium consists of Gram positive, anaerobic, spore forming bacilli. • Spores – wider than bacillary bodies – swollen appearance – resembling spindle. • ‘Kloster’ – spindle (Clostridium) • Causes three major diseases to humans – • Gas gangrene • Tetanus & • Food poisoning INTRODUCTION • Pathogens – C.perfringens, C.tetani – found normally in human & animal intestines • Many species – pathogenic but most are saprophytes – found in soil, water & decomposing plant & animal matter • Intestinal clostridia rapidly invade the blood & tissues of the host after death & initiate decomposition of the cadaver • C.acetobutylicum – industrial importance – production of chemicals like acetone & butanol MORPHOLOGY • Clostridia – highly pleomorphic • Rod shaped, usually 3 – 8 x 0.4 – 1.2 µm in size • Long filaments & involution forms – common • Spore formation occurs with varying frequency in different species – (C.sporogenes sporulate readily – C.perfringenes inconstantly) MORPHOLOGY • Shape & position of spores vary in different species – useful for identification & classification of clostridia – • Central or equatorial – spindle shaped (C.bifermentans) • Subterminal – club shaped (C.perfringens) • Oval & terminal – tennis racket (C.tertium) • Spherical & terminal – drumstick appearance (C.tetani) • Clostridia – motile with peritrichate flagella (exceptions – C.perfringens, C.tetani type VI) • Motility slow – described as ‘stately’ motility CULTURAL CHARACTERISTICS • C.perfringens & C.butyricum – capsulated (others are not) • Clostridia – easily stained – Gram positive (old cultures – Gram variable) • Clostridia – anaerobic (Sensitivity to oxygen varies in different species) • C.novyi – exacting anaerobe (die on exposure to oxygen) • C.histolyticum – aerotolerant (grow aerobically) CULTURAL CHARACTERISTICS • More important than absence of oxygen – provision of sufficiently low redox potential (Eh) in the medium • Achieved by adding reducing substances – • unsaturated fatty acids, • ascorbic acid, • glutathione, • cysteine, • thioglycolic acid, • alkaline glucose, • sulphites or • metallic iron Small concentration of CO2 enhances the groth CULTURAL CHARACTERISTICS • Optimum temperature 37°C (pathogenic clostridia) • Saprophytic clostridia – thermophilic & others psychrophylic • Optimum pH 7 – 7.4 • Growth – slow on solid media – colony characteristics variable • Some species are hemolytic on BA • Very useful medium – Robertson’s cooked meat medium CULTURAL CHARACTERISTICS • RCM contains – • unsaturated fatty acids (take up oxygen – reaction being catalysed by hematin in meat) • Sulphydril compounds – reduced Eh • Clostridia grow – making broth turbid • Gas produced by most species • Saccharolytic species – turn meat pink • Proteolytic species – turn meat black (produce foul & preservative odour) • In Litmus milk medium – production of acid, clot & gas can be detected RESISTANCE • Vegetative cells of clostridia – no difference from nonsporing anaerobes in their resistance to physical & chemical agents • Spores – exhibit variable resistance to heat, drying & disinfectants • Spores of C.botulinum survive boiling after 3 – 4 hrs & even at 105°C – not killed completely in less than 100 mts • Spores of many strains of C.perfringens – destroyed by boiling < 5 mts (some type A strains survive for several hrs) • C.tetani spores persist for years in dried earth • Spores of some strains of C.tetani – resist boiling for 15 – 90 mts • All species – killed by autoclaving at 121°C within 20 mts RESISTANCE • Spores – particularly resistant to phenolic disinfectants • Formaldehyde – not very active & spores sometimes survive in 2% solution for up to 5 days • Halogens – effective in 1% aqueous iodine sol. – kills spores within 3 hrs • Glutaraldehyde (2% at pH 7.5 – 8.5) – very effective in killing spores • Clostridia – susceptible to Metronidazole, Penicillin, Cephalosporins & Chloramphenicol; less to tetracyclines & resistant to aminoglycosides & quinolones • They produce disease – when conditions are appropriate • Invasive powers – limited (pathogenic clostridia form powerful exotoxins) RESISTANCE • C.botulinum – noninvasive & noninfectious • Botulism – due to ingestion of preformed toxin in food • C.tetani – little invasive property – confined to primary site of lodgement • Tetanus – due to action of potent exotoxin produced by it • Gas gangrene clostridia – being toxigenic also invasive – spread along tissues & even cause septicemia • Methods adopted for classification of clostridia – morphological features, shape & position of spores & biochemical features like saccharolytic & proteolytic capacities • Clostridia of medical importance – classified according to diseases they produce Classification based on Proteolysis Based on Human Pathogens CLOSTRIDIUM PERFRINGENS (C.welchii, Bacillus aerogenes capsulatus, B.phlegmonis emphysematosae) HISTORY & HABITAT • Originally cultivated by Achalme (1891) • First described in detail by Welch & Nuttall (1892) – isolated from blood & organs of cadaver • Most important of clostridia – causing Gas gangrene • Produces food poisoning & necrotic enteritis in human beings & many serious diseases in animals • Normal inhabitant of large intestines of human beings & animals • Found in feces & contaminates skin of perineum, buttocks & thigh • Spores – found in soil, dust & air MORPHOLOGY • Plump Gram positive bacillus with straight, parallel sides & rounded or truncated ends 4 - 6 x 1µm, occuring singly or in chains or small bundles • Pleomorphic, filamentous & involution forms common • Capsulated & nonmotile • Spores – central or subterminal (rarely seen in artificial cultures or from pathogenic lesions) • Their absence – characteristic morphological feature of C.perfringens CULTURAL CHARACTERISTICS • Anaerobe – also grows in microaerophilic conditions • pH range 5.5 – 8.0 • Temperature range 20 - 50°C (37°C) but 45°C optimal to many strains (useful for isolating pure cultures in mixtures) • Generation time – 10 mts • RCM – inoculated with mixtures of C.perfringens & other bacteria – incubated at 45°C for 4 – 6 hrs – only C.perfringens grows • Subcultures from this on BA yield pure & predominant growth of C.perfringens • Meat is turned to pink (not digested) • Culture has acidic reaction & sour odour CULTURAL CHARACTERISTICS • Litmus milk medium – • Fermentation of lactose leads to formation of acid (colour of litmus turns from blue to red) • Acid coagulates the casein (acid clot) • Clotted milk – disrupted due to vigorous gas production • Paraffin plug – pushed up & shreds of clot seen sticking to sides of the tube (Stormy fermentation) • After overnight incubation on Rabbit, Sheep or Human BA – colonies show • ‘Target Hemolysis’ (narrow zone of complete hemolysis due to theta toxin & • much wider zone of incomplete hemolysis due to alpha toxin) • Double zone pattern of hemolysis – fades on longer incubation BIOCHEMICAL REACTIONS & RESISTANCE • Glucose, maltose, lactose & sucrose – fermented with production of acid & gas • Indole – Negative • Methyl Red – Positive • VP – Negative • H2S – formed abundantly • Reduce nitrates • Spores – destroyed within 5 mts by boiling • Food poisoning strains of Type A & certain Type C strains resist boiling for 1 – 3 hrs • Autoclaving of 121°C for 15 mts – lethal • Spores – resistant to antiseptics & disinfectants in common use CLASSIFICATION • C.perfringens strains – classified into 5 types A to E – based on toxins they produce • Typing depends on ‘four major toxins’ – though it produces many toxins • Typing done by neutralization tests with specific antitoxins by • intracutaneous injections in guinea pigs or • intravenous injection in mice TOXINS & OTHER SUBSTANCES • C.perfringens – produces at least 12 distinct toxins, many enzymes & biologically active soluble substances • 4 major toxins – • alpha, • beta, • epsilon & • iota ALPHA TOXIN • Produced by all types of C.perfringens • Most abundantly by Type A strains • Most important toxin biologically • Responsible for profound toxemia of gas gangrene • Lethal, dermonecrotic & hemolytic Phospholipidase or • Lecithin ---------- Phosphoryl choline + diglyceride Lecithinase C Seen as an opalescence in serum or egg yolk media (neutralized by antitoxin) NAGLER REACTION • C.perfringens grown on medium containing – • 6% agar • 5% Fildes’ peptic digest of sheep blood • 20% human serum • Antitoxin spread on one half of plate • Colonies on other half without antitoxin – surrounded by a zone of opacity • No opacity around the colonies on half of the plate with antitoxin – due to specific neutralization of the alpha toxin • Specific lecithinase effect – Nagler Reaction – useful test for rapid detection of C.perfringens in clinical specimens. NAGLER REACTION Incorporation of Neomycin sulphate in medium – more selective Human serum – replaced by 5% egg yolk ALPHA TOXIN • Alpha toxin – hemolytic for RBC’s of most species, except horse & goat – due to its action on phospholipids on erythrocyte membranes • Lysis of hot-cold variety – best seen after incubation at 37°C followed by chilling at 4°C • Relatively heat stable toxin – partially inactivated by boiling for 5 mts. OTHER TOXINS • Beta (β), Epsilon (ε) & iota (ι) toxins – lethal & necrotizing properties • Gamma & eta toxins – minor lethal actions • Delta toxin – lethal & hemolytic for RBC’s of toed ungulates (sheep, goats, pigs & cattle) • Theta toxin – an oxygen labile hemolysin antigenically related to Streptolysin O (lethal & cytolytic toxin) • Kappa toxin – collagenase action • Lambda toxin – proteinase & gelatinase action • Mu toxin – hyaluronidase action • Nu toxin – deoxytribonuclease action ENZYMES & BIOLOGICALLY SOLUBLE SUBSTANCES • Enzymes which destroy blood group substance A & H • Neuraminidase – destroys myxovirus receptors on RBC • Substances – render RBC’s panagglutinable by exposing their T antigens • Hemagglutinin – active against RBC’s of human beings & most animals • Fibrinolysin • Hemolysin • Histamine • Bursting factor – specific action on muscle tissue (characteristic muscle lesions in ‘gas gangrene’) • Circulating factor – cause an increase in adrenaline sensitivity of capillary blood (inhibit phagocytosis) PATHOGENICITY • Human infections by C.perfringenes:• Gas gangrene • Food poisoning • Gangrenous appendicitis • Necrotizing enteritis • Biliary tract infection • Endogenous gas gangrene of intraabdominal origin • Brain abscess & Meningitis • Panophthalmitis • Thoracic infections • Urogenital infections GAS GANGRENE • C.perfringenes Type A – predominant agent • Occurs as sole agent but commonly seen in association with other clostridia & other anaerobes • All clostridial wound infections do not result in gas gangrene (wound contamination or anaerobic cellulitis) • Only if muscle tissues involves – gas gangrene results (anaerobic myositis) FOOD POISONING • Type A strains some times produce food poisoning • Characterized by – marked heat resistance of their spores & feeble production of alpha & theta toxins • Produce heat labile enterotoxin – like enterotoxins of V.cholerae & enterotoxigenic E.coli (fluid accumulation in rabbit ileal loop) • Caused by cold or warmed up meat dish FOOD POISONING • When contaminated meat cooked – spores in interior may survive • During storage or rewarming they germinate & multiply in anaerobic conditions in cooked meat • Many clostridia pass unharmed by gastric acid (due to high protein in meal) – reach intestines – produce enterotoxin • After incubation period of 8 – 24 hrs – abdominal pain, diarrhoea & vomiting sets up (Recovery occurs in 24 – 48 hrs) • Diagnosis – isolating heat resistant C.perfringens Type A from feces & food GANGRENOUS APPENDICITIS • C.perfringens Type A (occasionally type D) strains – isolated from gangrenous appendicitis • Demonstration of antitoxin & beneficial effects of administration of antitoxin – suggests etiological role of bacillus • Toxemia & shock of intestinal obstruction & peritonitis (some cases) – due to toxins of C.perfringens NECROTIZING ENTERITIS • Severe & fatal enteritis with different names – • Darmbrand (Germany) • Pigbel (New Guinea) • Caused by C.perfringens type C strains – with heat resistant spores – germinate in intestine producing beta toxin – mucosal necrosis • Immunization with type C toxoid – protects OTHERS • Biliary tract infection – • Acute emphysematous cholecystitis • Post cholecystectomy septicemia • Endogenous Gas gangrene of Intraabdominal origin – • Endogenous infection – contaminating abdominal wall during surgery • Brain abscess & meningitis • Rare OTHERS • Panophthalmitis – • Followed by penetrating injury • Thoracic infections – • Follow penetrating wounds of thorax • Urogenital infections – • Follow surgical procedure (nephrectomy) • Uterus – septic abortion SPECIES ASSOCIATED WITH GAS GANGRENE • Clostridium septicum • Clostridium novyi • Clostridium histolyticum GAS GANGRENE • Definition – Rapidly spreading edematous myonecrosis • Occuring characteristically in association with severe wounds of extensive muscle masses – contaminated with pathogenic clostridia particularly with C.perfringens • “Malignant edema” – in the past • “Anaerobic myositis” and “Clostridial myonecrosis” • Disease of war – extensive wounds with heavy contamination – common • Normal life – due to road accidents, crush injuries • Sometimes – single clostridium can cause this disease GAS GANGRENE • C.perfringens – frequently encountered (60%) • C.novyi • C.septicum 20 – 40 % • C.histolyticum – less common • Other clostridia – C.sporogenes, C.fallax, C.bifermentans, C.sordelli, C.aerofoetidum & C.tertium Implanted foreign particles (soil, road dust, bits of cloth) Endogenous (intestine) Clostridia Wounds Clostridia Iatrogenic (surgeries) Mere presence of clostridia in wound does not cause gas gangrene GAS GANGRENE • Mac Lennan – distinguished three types of anaerobic wound infections: • Simple wound – contamination with no invasion of underlying tissue (delayed wound healing) • Anaerobic cellulitis – Clostridia invade fascial planes with minimal toxin production & no invasion of muscle tissues • Gradual in onset – vary from ‘gas abscess’ to extensive involvement • Anaerobic myositis or gas gangrene – most serious, associated with clostridial invasion of healthy muscle tissues & abundant formation of exotoxins Only if favourable conditions of clostridial multiplication exists Battle wounds (implanted bullets, Shell fragments, bits of cloth) Crushing tissue (tearing of arteries) Anoxia of muscle Damage of tissue Outflow of blood Increase in press. Ionised calcium salts, Silicic acid necrosis Ideal conditions for growth of anaerobes Eh, pH Breakdown of carbohydrates Aminoacids from proteins GAS GANGRENE • Extravasated Hb & myoglobin – reduced & stop oxygen carriage • Aerobic oxidation stops & anaerobic reduction of pyruvate to lactate – fall in Eh • Clostridia multiply & elaborate toxins – tissue damage • Lecithinases damage cell membranes & increase capillary permeability – extravasation & increased tension in affected muscles – anoxic damage • Hemolytic anemia, hemoglobinuria – lysis of RBC’s by alpha toxin • Collagenases – destroy collagen barriers in tissues • Hyaluronidases – break intercellular substances – invasive spread GAS GANGRENE • Abundant production of gas – reduces blood supply by pressure effects – extending area of anoxic damage • Thus makes the lesion progressive • Incubation period – 7 hrs to 6 weeks after wound • 10 – 48 hrs – C.perfringens • 2 – 3 days – C.septicum • 5 – 6 days – C.novyi CLINICAL FEATURES • Increasing pain, tenderness & edema of affected part • Systemic signs of toxemia • Thin watery discharge from the wound – later profuse & serosanguinous • Accumulation of gas – tissues crepitant • Spreads rapidly in untreated cases • Profound toxemia & prostation – leading to death (circulatory failure) LAB DIAGNOSIS Confirmatory Primary 1. Clinical diagnosis Signs & symptoms GAS GANGRENE 2. Lab. Diagnosis Gram’s stain Anaerobic culture LAB DIAGNOSIS • Specimens to be collected:• Films from the muscles at the edge of the affected area • From the tissues in the necrotic area • From the exudate in the deeper parts of wound • Exudates from the parts – infection appears to be most active & from depths of wound (collected with capillary pipette or swab) • Necrotic tissue & muscle fragments GRAM’S STAIN • Differentiates anaerobic streptococcal myositis from gas gangrene • First – large number of streptococci with plenty of pus cells • Second – Diverse bacterial flora with scanty pus cells • Large number of regularly shaped Gram positive bacilli without spores – strongly suggests C.perfringens infection • “Citron bodies” & “Boat or leaf” shaped pleomorphic bacilli – irregular staining – C.septicum • Large bacilli with oval / subterminal spore – C.novyi • Slender bacilli – round, terminal spores – C.tetani GRAM’S STAIN CULTURE • Aerobic & anaerobic cultures – fresh or heated BA (5 – 6% agar) • Nagler’s reaction • Four tubes of RCM broth – inoculated, heated at 100°C for 5, 10, 15 & 20 mts – incubated & subcultured on BA plates after 24 – 48 hrs (differentiating organisms from heat resistant spores) • Isolates – identified based on their morphological, cultural, biochemical & toxigenic characters PROPHYLAXIS & THERAPY • Surgery – most important prophylactic & therapeutic measure • All damaged tissues – removed, wounds cleaned to remove blood clots, necrotic tissue & foreign material • Hyperbaric oxygen – beneficial • Antibiotics – effective in prophylaxis along with surgery • DOC – Metronidazole IV before surgery 8 hrly for 24 hrs • Broad spectrum antibiotic combination for both mixed aerobic & anaerobic is always useful – • Metronidazole • Gentamicin • Amoxycillin • Passive immunization – ‘anti-gas gangrene serum’ – rarely used Tetanus patient CLOSTRIDIUM TETANI INTRODUCTION Cl. tetani causes neurological disorder – Tetanus Cl. tetani produces a powerful neurotoxic exotoxin – Tetanospasmin - disrupts nerve impulses to muscles - mediates generalized muscle spasms Tetanus is characterized by increased muscle tone & spasms – common in nonimmunised persons & persons inadequately covered by immunisation Tetanus occurs in three clinical forms – generalized, neonatal & localized Cl. tetani widely distributed in soil, inanimate environment & intestines of human beings & animals Ubiquitous distribution – recovered from street & hospital dust, cotton wool, plaster of paris, bandages, catgut, talc, wall plaster & clothing Occurs as a harmless contaminant in wounds HISTORY Carle & Rattone (1884) – experimental transmission of disease to Rabbits Nicolaier (1884) – clinical manifestations of tetanus due to strychnine like poison Rosenbach (1886) – demonstrated slender bacillus with round terminal spores in a case of tetanus Kitasato (1889) – isolated bacillus in pure culture MORPHOLOGY Gram positive, slender bacillus with spherical terminal & bulging spores – Drumstick appearance Size – 4 to 8µm X 0.5 µm Noncapsulated & motile except Cl. tetani type VI with peritrichate flagella Young cultures are strongly gram positive & old cultures show variable staining Young spores are oval rather than spherical CULTURAL CHARACTERISTICS Obligate anaerobe Culture media – grows on ordinary media – growth is enhanced by addition of blood or serum Grows well on Robertson's cooked meat broth (RCMB), thioglycollate broth, NA & BA Optimum temp – 370C Optimum pH – 7.4 RCMB – turbidity, & some gas formation – meat not digested & is turned black on prolonged incubation CULTURAL CHARACTERISTICS Deep agar shake cultures -– spherical fluffy balls, 1-3 mm in diameter – filaments with radial arrangement BA – swarming – thin spreading fine translucent film of growth which is practically invisible except at the advancing edges Horse BA – α hemolytic colonies – β hemolytic due to tetanolysin (hemolysin) Gelatin stab cultures – fir tree type of growth Fildes technique – water of condensation at the bottom of a slope of NA inoculated with mixed culture – incubated anaerobically for 24h. – subcultures from the top of the tube – yield pure growth of Cl. tetani CLOSTRIDIUM TETANI ON BLOOD AGAR BIOCHEMICAL REACTIONS Weakly proteolytic & no saccharolytic property Doesn't ferment any sugar Indole positive MR & VP negative H2S not formed Nitrates not reduced Greenish fluorescence produced on media containing neutral red (MA) RESISTANCE Strains are killed by boiling for 10 – 15 min. Some strains resist boiling for 3 hrs. Autoclaving (121 0C for 20 min.) kills the spores of most strains Spores are killed in few hrs. by common disinfectants - 1% aqueous solution of Iodine, 10 volumes of Hydrogen peroxide & 2% glutaraldehyde Spores survive is soil for years & resistant to most antiseptics - 5% phenol or 0.1% mercuric chloride ANTIGENIC STRUCTURE & TOXINS Based on type specific flagellar Ags – 10 serological types – I to X All the types produce same toxin & neutralized by antitoxin produced against any one type Tetanolysin – hemolysin Tetanospasmin – powerful hemotoxin TETANOLYSIN Heat labile & oxygen labile Antigenically related to oxygen labile hemolysins of Cl. perfringes, Cl. novyi & St. pyogenes Causes lysis of erythrocytes – rabbit & horse species Leucotoxic TETANOSPASMIN Heat labile & oxygen stable, inactivated at 65 0C in 5 min. Powerful neurotoxin & rapidly destroyed by proteolytic enzymes Plasmid coded & protein in nature & neutralised by antitoxin Responsible for clinical manifestations of tetanus TETANOSPASMIN Lethal to mice & guinea pigs in minute doses (0.0000001 mg for mouse) Can be toxoided – spontaneously or with formaldehyde On release from bacillus, toxin gets autolysed chain & light chain joined by disulphide bond heterodimer consisting of heavy M.W – 150,000 – heavy chain mediates binding to nerve cell receptors – light chain blocks neurotransmitter release at NMJ PATHOGENESIS Cl. tetani is not an invasive organism Contamination of wound with Cl. tetani spores Tetanus Source of infection – soil, dust & faeces etc. Infection remains localised in the wound Favourable conditions for germination of spores & Toxin production Reduced O-R potential Devitalised tissues Foreign bodies Concurrent infection PATHOGENESIS Tetanospasmin pathogenic effects Toxin produced locally absorbed by motor nerve endings CNS Toxin is specifically fixed by gangliosides of grey matter of nervous tissue Effects of Tetanospasmin resembles strychnine Tetanus toxin specifically blocks synaptic inhibition in spinal cord Toxin acts presynaptically Abolition of spinal inhibition uncontrolled spread of impulses initiated any where in CNS muscle rigidity & spasms due to simultaneous contraction of agonists & antagonists CLINICAL MANIFESTATIONS (GENERAL TETANUS) Toxin enters lymphatics & blood stream – spreads to distant nerve terminals Sequential involvement of nerves of head, neck, chest, trunk & extremities – hands & feet are spared Characterized by increased muscle tone & generalized spasms Onset is usually 7 – 14 d. after injury Course of the disease – 4 – 6 wks. Recovery is usually complete Pt. first notices increased tone in masseter muscles – Trismus or Lock Jaw GENERAL TETANUS Dysphagia or stiffness or pain in the neck, shoulder & back muscles Subsequent involvement of other muscles produce rigid abdomen & stiff proximal limb muscles Sustained contraction of facial muscles – grim face or sneer – Risus sardonicus Contraction of back muscles – arched back – Opisthotonos Paroxysmal, voilent, painful, generalized muscle spasms – cyanosis & threaten ventilation – laryngo spasm GENERAL TETANUS Autonomic dysfunction – hypertension, tachycardia, profuse sweating, peripheral vasoconstriction etc. Complications – aspiration pneumonia, fractures, muscle rupture, pulmonary emboli, decubitus ulcers, deep vein thrombophlebitis etc. Pt. usually requires prolonged ventilatory support CLINICAL FEATURES Localized tetanus Uncommon – restricted to muscles near the wound – muscle rigidity & few spasms Cephalic tetanus Rare form of local tetanus Follows head injury or ear infection – trismus & dysfunction of one or more crainal nerves – mortality is very high NEONATAL TETANUS Commonly seen in neonates born to inadequately immunized mothers or Unsterile treatment of umbilical cord stump Onset – within first two weeks of life Poor feeding, rigidity & spasms are typical features EFFECTS OF TETANOSPASMIN BY DIFFERENT ROUTES OF ADMNISTRATION Oral route destroyed by digestive enzymes – No effect S/C, I/M & IV injections are equally effective Intraneural injections are more lethal Route of administration modifies clinical picture Experimental tetanus manner in which toxin reached & disseminated in CNS localized, Ascending or descending types ANIMAL PATHOGENECITY Horse is most susceptible to tetanus toxin Guinea pigs > mice > goats > rabbits are susceptible Birds & reptiles are resistant ANIMAL PATHOGENICITY I/M inoculation of toxin into one of the hind limbs Toxin acts on the segment of the spinal cord containing motor neurons of the nerve supplying inoculated area tonic spasms of muscles of inoculated limb – Local Tetanus Spread of toxin up the spinal cord Ascending Tetanus – opposite hindlimb, trunk & forelimbs involved in an ascending fashion I/V inoculation of toxin Descending tetanus - spasticity develops first in muscles of the head & neck & spreads downwards EPIDEMIOLOGY Tetanus is a life threatening disease with high mortality 80-90% in untreated cases In treated cases case fatality rate varies 15 – 50% Common in developing countries 80% cases occur in Africa & S.E Asia – common infectious disease causing death particularly in neonates Common in places where unhygienic practices are common especially rural India Warm climates, summer months EPIDEMIOLOGY Neonatal tetanus – occurs from contamination of umbilical stump – traditional practices such as cutting umbilical cord with bamboo & applying soil, cowdung or engine oil etc. Ritual surgery such as ear piercing or circumcision may also cause infection Malaria & HIV in mother reduces placental transfer of maternal antibody tetanus neonatal Tetanus following I/M injections is common especially with quinine – low pH of the drug facilitates toxin entry into nerves local necrosis EPIDEMIOLOGY I.P is variable (6-12d) – influenced by various factors – site & nature of wound – dose & toxigenicity of organism & immune status of pt. Short I.P – grave prognosis Common among males Tetanus is characterized by tonic muscular spasms at the site of infection generalised involving whole of the somatic muscular system EPIDEMIOLOGY Disease usually follows : Injury – punctured wounds, laceration or abrasions Surgical operations – due to lapses in asepsis Local suppuration – otitis media (otogenic tetanus) Septic abortion Unsterile injections Chronic conditions – abscesses, gangrene, skin ulcers, burns, frostbite & drug abuse LABORATORY DIAGNOSIS Primary Clinical Diagnosis Signs & symptoms Confirmatory Diagnosis Microbiological Diagnosis Demonstration of Cl. tetani bacilli LABORATORY DIAGNOSIS Culture Animal inoculation Microbiological Diagnosis Demonstration of Cl. tetani bacilli Microscopy SPECIMEN COLLECTION Wound swabs Exudate Excised bits of tissue from necrotic depths of wounds SPECIMEN TRANSPORTATION PROCESSING OF SPECIMENS (MICROSCOPY) Gram staining of primary smears Gram positive bacilli with drum stick appearance – indistinguishable from Cl. tetanomorphum & Cl. sphenoides CULTURE Isolation of Cl. tetani from clinical specimens Culture media – BA & RCMB Clinical material One half of BA. Plate Plates incubated at 370C anaerobically for 24-48 h. 3 tubes of RCMB Heated at 800C for 15 min. Heated at 800C Unheated for 5 min. Incubated at 370C for 24-48 h. RCMB 800c 15 min. 800c 5 min. Unheated Heating leads to death of vegetative forms and not spores CONTD… S/C daily on one half of BA for 4d. Swarming growth of Cl. tetani detected on opposite half of plate Gram stain smear of colonies Typical drum stick appearance Addition of Polymyxin B – makes the BA plate more selective TOXIGENICITY TESTING • Invitro – in Laboratory • Invivo – in Animal INVITRO • Blood agar plates (with 4% agar – to inhibit swarming) – having tetanus antitoxin (1500 units/ml) spread over ½ of plate • Toxigenic strains inoculated and incubated anaerobically for 2 days • Cl.tetani toxigenic strains show hemolysis on the side where antitoxin is absent • Not reliable as it indicates on tetanolysin and not tetanospasmin INVIVO Demonstration of Toxin production by the organism isolated from clinical specimens 0.2 ml of 2-4 d old cooked meat culture inoculated into 2 mice (root of tail of mouse) Test animal Control animal Receives tetanus antitoxin (1000 units) an hour earlier Symptoms develop Within 24-48h. • stiffness of tail rigidity proceeds to the leg on the inoculated side another leg trunk fore limbs death in 2d. No symptoms Due to neutralization of toxin by Antitoxin PROPHYLAXIS Tetanus is a preventable disease Objective – to build up antitoxic immunity by active immunisation Prophylaxis depends on the type of wound & immune status of the patient PROPHYLAXIS Surgical Antibiotics Immunisation Surgical prophylaxis Removal of foreign body & blood clots – to prevent anaerobic conditions Simple cleansing of wound to radical excision of tissue IMMUNIZATION (ACTIVE) Prompt administration of antibiotics Destroy or inhibit tetanus bacilli & other pyogenic bacteria in wounds Penicillin or erythromycin are the drugs of choice – to be started before wound toilet Bacitracin or neomycin applied locally Antibiotics have no action on toxin Antibiotics serve as adjunct to immunisation ACTIVE IMMUNIZATION Full course of three doses confers immunity for 10 yrs. Booster dose of toxoid recommended after 10 yrs. ATS or TIG should not be given to an immunised individual Booster dose of toxoid given if wounding occurs 3 yrs. or more after full course of immunisation ACTIVE IMMUNIZATION Tetanus toxoid given with triple vaccine DPT- pertussis acts as an adjuvant – 3 doses given I/M at interval of 4-6 wks. – starting at age as early as 6 wks Booster doses given at 18 mths. & 5 yrs. (DT) PASSIVE IMMUNIZATION Reserved as Emergency procedure – given only once Antitetanus serum (ATS) – 1500 IU given immediately by I/M route after wounding Prior to administration of ATS skin test to be done (0.5 ml S/C) in hypersensitive individuals Human antitetanus Ig (HTIG) – 250 U given I/M advantages – no hypersensitivity reactions as it is homologous serum prepared from humans COMBINED PROPHYLAXIS Nonimmune persons – emergency procedure First dose of TT in one arm along with administration of ATS or HTIG in another arm Second & third dose of TT given at monthly interval PROPHYLAXIS OF TETANUS IN THE WOUNDED TREATMENT Person to person transmission does not occur & tetanus is not infectious Pts. to be treated in isolated units Pts. to be protected from noise & light which may provoke convulsions Controlling of spasms, maintaining airway & attention to feeding Antibiotic therapy with Penicillin or Metronidazole to be started at once & continued for a wk. or more Pts. recovering from tetanus should be immunised with full course of tetanus toxoid as an attack of tetanus does not confer immunity Human TIG 10,000 IU given by slow IV infusion followed by 5000 IU if needed – inactivates unbound toxin & any further toxin that maybe produced Other CLOSTRIDIA Pathogenic clostridia • C.perfringenes • C.histiolyticum • C.tetani • C.botulinum • C.septicum • C.difficile • C.novyi Clostridium septicum • First described by Pasteur & Joubert (1887) – as Vibrion septique • Pleomorphic, 3 – 8 x 0.6µm in size, oval, central or subterminal spore • Motile with peritrichate flagella • Grows anaerobically on ordinary media • Colonies – irregular & transparent initially, turning opaque on continued incubation • Hemolysis – on horse blood agar • Growth promoted by glucose • Saccharolytic & produces abundant gas …contd… • Six groups – based on somatic & flagellar antigens • C.septicum – produces at least four distinct toxins & a fibrinolysin • Alpha toxin – hemolytic, dermonecrotic & lethal • Beta toxin – leucotoxic deoxyribonuclease • Gamma toxin – hyaluronidase • Delta toxin – an oxygen labile hemolysin • C.septicum – found in soil or animal intestines • Associated with gas gangrene in human beings • Also causes ‘Braxy’ in sheep & ‘Malignant edema’ in cattle & sheep Clostridium novyi • Large, stout, pleomorphic, Gram positive bacillus with large, oval, subterminal spores • Widely distributed in soil • Strict anaerobe, readily inactivated by exposure of cultures to air • Four types (A to D) – based on production of toxins • Only type A – medically important – causing gas gangrene • Gas gangrene – by C.novyi – characterized by high mortalilty & large amounts of edema fluid with little or no observable gas in infected tissue • A lethal outbreak of C.novyi type A infection in heroin addicts in Britain occurred in 2000. Clostridium histiolyticum • An actively proteolytic clostridium, forming oval, subterminal, bulging spores • Aerotolerant & some growth occurs even in aerobic cultures • It forms at least five distinct toxins • It is infrequently associated wit gas gangrene in humans Anaerobic myositis Necrotizing enteritis Clostridium botulinum INTRODUCTION & HABITAT • C.botulinum – causes botulism, a paralytic disease usually presenting as a form of food poisoning • Botulism – name derived from – ‘Sausage’ (botulus in Latin) – associated with this type of food poisoning • Fist isolated by van Ermengem (1896) – from a piece of ham – caused an outbreak of botulism • Bacillus – widely distributed saprophyte – in virgin soil, vegetables, hay, silage, animal manure & sea mud MORPHOLOGY • Gram positive bacillus • 5 x 1µm in size • Non capsulated • Motile by peritrichate flagella • Subterminal or oval bulging spores CULTURAL CHARACTERISTICS • Strict anaerobe • Optimum temp. 35°C – some strains grow at 1 – 5°C • Good growth occurs in ordinary media • Surface colonies – large, irregular, semitransparent with fimbriate border • Biochemical reactions vary in different types • Spores – produced consistently when grown in alkaline glucose gelatin media at 20 - 25°C • Not usually produced at higher temperatures RESISTANCE • Spores – heat & radiation resistant • Spores - survive for several hours at 100°C & up to 10 minutes at 120°C • Spores of non proteolytic types of B, E & F – much less resistant to heat CLASSIFICATION • Eight types of C.botulinum – identified based on immunological difference in toxins produced by them • Type A, B, C1, C2, D, E, F & G • Toxins produced by different types – identical in their pharmacological activity but neutralized only by their homologous antiserum • Only C2 – enterotoxic activity (all others are neurotoxins) TOXIN • C.botulinum produces – powerful exotoxin that is responsible for its pathogenicity • Toxin differs from other exotoxins – it is not released during life of the organism • Produced intracellulary & appears in the medium only after death & autolysis of the cell • It is believed to be synthesized initially as a nontoxic protoxin or progenitor toxin • Trypsin & other proteolytic enzymes activate progenitor toxin to active toxin • Toxin – isolated as a pure crystalline protein – probably most toxic substance …contd… • Molecular weight – 70,000 & lethal dose for mice of 0.000000033mg • Lethal dose for human beings 1 - 2µg • Neurotoxin & acts slowly, taking several hours to kill • Toxin is relatively stable, being inactivated only after 30 – 40 mts at 80°C and 10 mts at 100°C • Food suspected to be contaminated with botulinum toxin – rendered completely safe by pressure cooking or boiling for 20 mts • Toxin resists digestion & absorbed through the small intestines in active form …contd… • Action – by blocking production or release of acetylcholine at the synapses & neuromuscular junctions • Onset – marked by diplopia, dysphagia & dysarthria due to cranial nerve involvement • Symmetric descending paralysis – characteristic pattern, ending in death by respiratory paralysis • Small quantity of C.botulinum type A toxin injected into a muscle – selectively weakens it by blocking the release of acetylcholine at the neuromuscular junction • Muscles – injected will atrophy – recovers in 2 – 4 months as new terminal axon sprouts & restores transmission …contd… • Intramuscular injection of toxin – first used to treat strabismus – now recognized as a safe & effective symptomatic therapy for many neuromuscular diseases • Botulinum toxin can be toxoided • Specifically neutralized by its antitoxin & is a good antigen • Toxins produced by different types of C.botulinum appear to be identical, except for immunological differences • Toxin production appears to be determined by – presence of bacteriophages, at least in types C & D PATHOGENICITY • C.botulinum – noninvasive & virtually noninfectious • Its pathogenicity is due to action of its toxin – manifestations of which are collectively called botulism • Botulism is of three types • Food borne Botulism • Wound Botulism & • Infant Botulism Food borne Botulism • Due to ingestion of preformed toxin • Types of bacillus & nature of food responsible vary in different regions • Human disease – usually caused by types A, B, E & very rarely F • Types C & D – usually associated with outbreaks in cattle & wild fowl • Type G – associated with sudden death in few patients • Source of botulism – usually preserved food – meat & meat products, canned vegetables & fish • Type E – associated with fish & other sea foods • Proteolytic varieties of C.botulinum – digest food – which appears spoiled later 12 - 36 …contd… • Cans often are inflated & show bubbles on opening • Non proteolytic varieties leave food unchanged • Symptoms begin usually 12 – 36 hrs after ingestion of food • Vomiting, thirst, constipation, ocular paresis, difficulty in swallowing, speaking & breathing constitute the common features • Coma or delirium may supervene • Death – due to respiratory failure & occurs 1 – 7 days after onset • Case fatality varies from 25 – 70 percent Wound Botulism • Very rare condition resulting from wound infection with C.botulinum • Toxin – produced at the site of infection is absorbed • Symptoms are of food borne botulism except for GI symptoms which are absent • Type A – responsible for most of the cases Infant Botulism • Toxic infection • C.botulinum spores – ingested in food – established in gut & there produce the toxin • Occur in infants below six months • Manifestations – constipation, poor feeding, lethargy, weakness, pooled oral secretions, weak or altered cry, floppiness & loss of head control • Patients excrete toxin & spores in their feces • Toxin – not demonstrable in blood …contd… • Management consists of supportive care & assisted feeding • Antitoxins & antibiotics – not indicated • Degrees of severity – very mild illness to fatal disease • Sometimes – sudden infant death syndrome occurs – due to infant botulism • Honey – likely food through which bacillus enters gut LABORATORY DIAGNOSIS • Confirmed by demonstration of • Bacillus or • Toxin Food & feces • Gram positive sporing bacilli – demonstrable in smears from food • C.botulinum – isolated from food or patients feces Gram’s stain of Clostridium botulinum Spore Specimens Infected food or feces Saline Symptoms Macerated food Control animal protected by polyvalent antitoxin – remains healthy …contd… • Typing done by passive protection with type specific antitoxin • Toxin – occasionally demonstrable in patient’s blood or in liver (postmortem) • Retrospective diagnosis – made by detection of antitoxin in patient’s serum CONTROL • Botulism follows consumption of inadequately canned or preserved food • Control achieved by proper canning & preservation • In an outbreak – prophylactic dose of antitoxin – given IM to all who consumed the contaminated food • Active Immunization – also effective • Two injections of aluminium sulphate adsorbed toxoid – given at an interval of 10 weeks – followed by a booster dose a year later • Antitoxin – tried for treatment • Polyvalent antiserum to types A, B & E may be administered – as soon as a clinical diagnosis is made • Supportive therapy – maintenance of respiration of equal or greater importance Clostridium difficile & Antibiotic associated colitis INTRODUCTION • First isolated in 1935 – from feces of new born infants • So named because – difficulty in isolating • Long, slender, Gram positive bacillus with pronounced tendency to lose its Gram reaction • Spores large, oval & terminal • Non hemolytic, saccharolytic & weakly proteolytic • Not considered pathogenic till 1977 – later found responsible for antibiotic associated colitis PATHOGENICITY • Acute colitis – with or without membrane formation – an important complication of oral antibiotic therapy • Many antibiotics are responsible • Ampicillin • Tetracycline • Chloramphenicol • Lincomycin • Clindamycin Prone for pseudomembranous colitis • Antibiotic associated colitis – due to active multiplication of C. difficile & its production of enterotoxin as well as cytotoxin DIAGNOSIS & TREATMENT • Demonstrating the toxin in feces of patients by its characteristic effect on • Hep – 2 & • Human diploid cell cultures or • by ELISA • Toxin – specifically neutralized by C.sordelli antitoxin • C.difficile – also grown from feces of patients • Strains – usually resistant to most antibiotics • DOC – Metronidazole • Vancomycin & Bacitracin – also useful
