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Corynebacteria II MBBS Dr Ekta Chourasia Microbiology


Gram positive rods Non spore-forming 1. AEROBIC 

Spore-forming 1. AEROBIC

Corynebacteria   

C. diphtheriae diphtheroids C. jeikeium

Genus: Bacillus   

Listeria monocytogenes  Erysipelothrix rusiopathiae

B. anthracis B. cereus B. subtilis

2.

ANAEROBIC 

2.

ANAEROBIC 

Genus: Clostridium  

Lactobacillus spp.

 

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C. tetani C. botulinum C. difficile C. perfringens


Corynebacteria - Overview 

Gram positive, non motile bacilli with irregularly stained segments

Frequently show club shaped swellings – corynebacteria (coryne = club)

C. diphtheriae : most important member of this genus, causes diphtheria

Diphtheroids : commensals of nose, throat, nasopharynx, skin, urinary tract & conjunctiva.

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Historical overview I. Corynebacterium diphtheriae Bretonneau 1826 Clinical characterisation of diphtheria – diphtherite Klebs 1883 Detecting the bacterium

Behring and Kitasato 1890-1892 - Discovering the diphtheria antitoxin - Antitoxic immunity (therapy and prevention)

Loeffler 1884 Isolating the bacterium

Roux 1894 Treatment with antitoxin

Roux and Yersin 1888 Discovering the diphtheria toxin

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Historical overview I. Corynebacterium diphtheriae Emil von Behring 1901 Nobel prize Behring 1913 Active immunisation I. with toxin-antitoxin mix

Ramon 1923 Active immunisation II. Anatoxin = toxoid Freeman 1951 PHAGE (lysogenia, toxin production)

Schick 1913 Skin test 14.12.08

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Introduction – C. diphtheriae 

Diphtheros – leather (tough, leathery pseudomembrane)

Also known as Klebs–Loeffler bacillus

Causes Diphtheria

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Important features of C. diphtheriae     

Slender Gram positive bacilli Pleomorphic, non motile, non sporing Chinese letter or Cuneiform arrangement Stains irregularly, tends to get easily decolorised May show clubbing at one or both ends Polar bodies/ Metachromatic granules/ volutin or Babes Ernst granules Metachromatic Granules: made up of polymetaphosphate  Bluish purple color with Loeffler’s Methylene blue  Special stains: Albert’s, Neisser’s & Ponder’s 

Grows aerobically at 37°C

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Virulence factor 

Exotoxin – Diphtheria toxin:  Protein

in nature  very powerful toxin  Responsible for all pathogenic effects of the bacilli  Produced by all the virulent strains  Two fragments A & B

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Diphtheria toxin – Mechanism of action DT - Acts by inhibition of protein synthesis

Fragment A – inhibits polypeptide chain elongation by inactivating 14.12.08

Dr Ekta, Microbiology the Elongation factor EF 2 in the presence of NAD


Diphtheria Toxin 

Toxigenicity can be induced by Lysogenic or phage conversion – corynephages (tox+ phage) or beta phages

Can be toxoided by 1. 2. 3. 4.

Prolonged storage Incubation at 37°C for 4 - 6 weeks Treatment with 0.2 – 0.4 % formalin or Acid pH.

Stain used for toxin production – ‘Park Williams 8’ strain

Antibodies to fragment B - protective

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Epidemiology 

Habitat – nose, throat, nasopharynx & skin of carriers and patients

Spread by respiratory droplets, usually by convalescent or asymptomatic carriers

Nasal carriers harbour the bacilli for longer time than pharyngeal carriers

Local infection of throat - toxemia

Incubation period of diphtheria – 3 to 4 days

In tropics, cutaneous infection is more common than respiratory infection

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Diphtheria 

Site of infection 1. 2. 3. 4. 5. 6. 7.

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Faucial (palatine tonsil) – commonest type Laryngeal Nasal Otitic Conjunctival Genital – vulval, vaginal, prepucial Cutaneous – usually a secondary infection on preexisting lesion, caused by non toxigenic strains

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Pathogenesis & Clinical Manifestations Human Disease

Usually begins in respiratory tract Virulent diphtheria bacilli lodge in throat of susceptible individual Multiply in superficial layers of mucous membrane Elaborate toxin which causes necrosis of neighboring tissue cells Inflammatory response eventually results in pseudomembrane (fibrinous exudate with disintegrating epithelial cells, leucocytes, erythrocytes & bacteria)

1. 2. 3. 4. 5.

  

Usually appears first on tonsils or posterior pharynx and spreads upward or down In laryngeal diphtheria, mechanical obstruction may cause suffocation Regional lymphnodes in neck often enlarged (bull neck)

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Diphtheria - Clinical Classification 

Based on the severity of clinical presentation: 1.

Malignant or hypertoxic – severe toxemia with marked adenitis

2.

Septic – ulceration, cellulitis, & gangrene around the pseudomembrane

3.

Hemorrhagic – bleeding from the edge of membrane, epistaxis, conjunctival hemorrahge, purpura & generalized bleeding tendency.

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Complications of diphtheria 

Mechanical complications are due to the pseudomembrane, while the systemic effects are due to the toxin. 1. 2. 3.

4.

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Asphyxia – due to obstruction of respiratory passage Acute circulatory failure Postdiphtheritic paralysis – occurs in 3rd or 4th week of disease, palatine & ciliary, spontaneous recovery Sepsis – pneumonia & otitis media

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Laboratory Diagnosis 

Specimen – swab from the lesions

1.

Microscopy   

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Gram stain: Gram +ve bacilli, chinese letter pattern Immunofluorescence Albert’s stain for metachromatic granules Dr Ekta, Microbiology


Laboratory Diagnosis 2.

Culture – isolation of bacilli requires media enriched with blood, serum or egg a. b. c. d. e.

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Blood agar Loeffler’s serum slope – rapid growth, 6 to 8 hrs Tellurite blood agar – tellurite is reduced to tellurium, gives gray or black color to the colonies Hoyle’s media modifications of TBA McLeod’s media

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Growth of diphtheria bacilli

Blood agar

Tellurite blood agar

Loeffler’s serum slope 14.12.08

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Biotypes of Diphtheria bacilli 

Based on colony morphology on the tellurite medium & other properties, McLeod classified diphtheria bacilli into three types: Features

1. Gravis

2. Intermedius

3. Mitis

Case fatality rate

High

High

Low

Complications

Paralytic, hemorrhagic

Hemorrhagic

Obstructive

Predominance

In epidemic areas Epidemic areas

Spread

Rapid

Rapidly than mitis Less rapid

Colony on TBA

‘Daisy head” colony

‘Frog’s egg colony

‘Poached egg’ colony

Hemolysis

Variable

Nonhemolytic

Usually hemolytic

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Endemic areas


Laboratory Diagnosis 3.

Biochemical reactions a.

Hiss's serum water - ferments sugar with acid formation but not Gas ferments: glucose, galactose, maltose and dextrin

b.

Resistant to light, desiccation and freezing

c.

Sterilization: sensitive to heat (destroyed in 10mins at 58째C or 1min in 100째C), chemical disinfectants

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Laboratory Diagnosis 4.

Virulence tests - Test for toxigenicity A.

Invivo tests – animal inoculation (guinea pigs) a. b.

A.

Invitro tests a. b.

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Subcutaneous test Intracutaneous test

Elek’s gel precipitation test Tissue culture test Dr Ekta, Microbiology


Laboratory Diagnosis Virulence tests - Invivo tests   I.

Bacterial growth from Loeffler’s serum slope is emulsified in 2-4 ml broth. Two guinea pigs (GP A and GP B) Subcutaneous test – 0.1 ml of emulsion is injected SC into each guinea pig GP A - has diphtheria antitoxin (500 units injected 18 to 24 hours before) GP B - Doesn't have antitoxin

I.

Intracutaneous test - 0.1 ml of emulsion is injected IC into each guinea pig GP A - has diphtheria antitoxin (500 units injected 18 to 24 hours before) GP B – 50 units of antitoxin IP four hrs after the skin test

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Laboratory Diagnosis Virulence tests - Invitro tests I.

Elek's gel precipitation test  

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filter paper saturated with antitoxin (1000units/ ml) is placed on agar plate with 20% horse serum bacterial culture streaked at right angles to filter paper

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Laboratory Diagnosis Virulence tests - Invitro tests II.

Tissue culture test - incorporation of bacteria into agar overlay of eukaryotic cell culture monolayers. Result: toxin diffuses into cells and kills them

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Treatment 

specific treatment must not be delayed if clinical picture suggests of diphtheria

rapid suppression of toxin-producing bacteria with antimicrobial drugs (penicillin or erythromycin)

early administration of antitoxin: 20,000 to 1,00,000 units for serious cases, half the dose being given IV

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Prophylaxis 1)

Active Immunization (Vaccination) i.

Formol toxoid (fluid toxoid)  

ii.

Adsorbed toxoid (more immunogenic than fluid toxoid)  

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incubation of toxin with 0.3% formalin at pH 7.4 - 7.6 at 37°C for 3 to 4 weeks fluid toxoid is purified and standardized in flocculating units (Lf doses)

purified toxoid adsorbed onto insoluble aluminium phosphate or aluminium hydroxide given IM (DTP or TD)

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Prophylaxis 

Adsorbed Toxoid a.

DPT - triple vaccine given to children; contains diphtheria toxoid, Tetanus toxoid and pertussis vaccine

b.

DaT - contains absorbed tetanus and ten-fold smaller dose of diphtheria toxoid. (smaller dose used to diminish likelihood of adverse reactions)

Schedule i) Primary immunization - infants and children - 3 doses, 4-6 weeks interval - 4th dose after a year - booster at school entry

ii) Booster immunization - adults -Td toxoids used (travelling adults may need more) 

SHICK test - to test susceptibility to vaccine, not done now-a-days

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Prophylaxis 2.

Passive immunization

3.

Combined immunization

ADS (Antidiphtheritic serum, antitoxin) made from horse serum - 500 to1000 units subcutaneously

First dose of adsorbed toxoid + ADS, to be continued by the full course of active immunisation

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CONTROL 1. 2. 3.

isolate patients treat with antibiotics actively complete vaccination schedule should be used with booster every 5 years

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Other Corynebacteria 

C. ulcerans – diphtheria like lesions in guinea pigs & cows, may get transmitted to humans by cow’s milk

Diphtheroids –     

Normal commensals of nose, throat, nasopharynx, skin, urinary tract & conjunctiva Stain uniformly Few or no metachromatic granules Arranged in parallel rows (palisades) Nontoxigenic

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