Corynebacteria II MBBS Dr Ekta Chourasia Microbiology

Corynebacteria

II MBBS

Dr Ekta ChourasiaMicrobiology

Dr Ekta, Microbiology14.12.08

Gram positive rods

Non spore-forming1. AEROBIC

Corynebacteria C. diphtheriae diphtheroids C. jeikeium

Listeria monocytogenes Erysipelothrix rusiopathiae

2. ANAEROBIC Lactobacillus spp.

Spore-forming1. AEROBIC

Genus: Bacillus B. anthracis B. cereus B. subtilis

2. ANAEROBIC Genus: Clostridium

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.


Historical overview I.Corynebacterium diphtheriae

Bretonneau 1826Clinical characterisationof diphtheria – diphtherite

Klebs 1883Detecting the bacterium

Loeffler 1884Isolating the bacterium

Roux and Yersin 1888Discovering the diphtheria toxin

Behring and Kitasato1890-1892- Discovering the diphtheria

antitoxin- Antitoxic immunity (therapy and

prevention)

Roux 1894Treatment with antitoxin


Historical overview I.Corynebacterium diphtheriae

Emil von Behring 1901

Nobel prize

Behring 1913

Active immunisation I.

with toxin-antitoxin mix

Schick 1913

Skin test

Ramon 1923

Active immunisation II.

Anatoxin = toxoid

Freeman 1951

PHAGE (lysogenia,

toxin production)


Introduction – C. diphtheriae

Diphtheros – leather (tough, leathery pseudomembrane)

Also known as Klebs–Loeffler bacillus

Causes Diphtheria


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


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


Diphtheria toxin – Mechanism of action

Fragment A – inhibits polypeptide chain elongation by inactivating

the Elongation factor EF 2 in the presence of NAD

DT - Acts by inhibition of protein synthesis


Diphtheria Toxin Toxigenicity can be induced by Lysogenic or phage

conversion – corynephages (tox+ phage) or beta phages

Can be toxoided by -1. Prolonged storage2. Incubation at 37°C for 4 - 6 weeks3. Treatment with 0.2 – 0.4 % formalin or4. Acid pH.

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

Antibodies to fragment B - protective


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


Diphtheria

Site of infection 1. Faucial (palatine tonsil) – commonest type2. Laryngeal3. Nasal4. Otitic5. Conjunctival6. Genital – vulval, vaginal, prepucial7. Cutaneous – usually a secondary infection on pre-

existing lesion, caused by non toxigenic strains


Pathogenesis & Clinical Manifestations Human Disease

1. Usually begins in respiratory tract 2. Virulent diphtheria bacilli lodge in throat of

susceptible individual 3. Multiply in superficial layers of mucous

membrane 4. Elaborate toxin which causes necrosis of

neighboring tissue cells 5. Inflammatory response eventually results in

pseudomembrane (fibrinous exudate with disintegrating epithelial cells, leucocytes, erythrocytes & bacteria)

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)



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.


Complications of diphtheria

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

1. Asphyxia – due to obstruction of respiratory passage

2. Acute circulatory failure 3. Postdiphtheritic paralysis – occurs in 3rd or 4th week

of disease, palatine & ciliary, spontaneous recovery

4. Sepsis – pneumonia & otitis media



Laboratory Diagnosis Specimen – swab from the

lesions

1. Microscopy Gram stain: Gram +ve bacilli,

chinese letter pattern Immunofluorescence Albert’s stain for

metachromatic granules


Laboratory Diagnosis

2. Culture – isolation of bacilli requires media enriched with blood, serum or egg

a. Blood agar

b. Loeffler’s serum slope – rapid growth, 6 to 8 hrs

c. Tellurite blood agar – tellurite is reduced to tellurium, gives gray or black color to the colonies

d. Hoyle’s media modifications of TBA

e. McLeod’s media


Growth of diphtheria bacilli

Blood agar

Loeffler’s serum slope

Tellurite blood agar


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 Endemic 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



3. Biochemical reactions a. Hiss's serum water - ferments sugar with acid

formation but not Gasferments: glucose, galactose, maltose and dextrin

b. Resistant to light, desiccation and freezing

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



4. Virulence tests - Test for toxigenicityA. Invivo tests – animal inoculation (guinea

pigs)a. Subcutaneous test

b. Intracutaneous test

B. Invitro testsa. Elek’s gel precipitation test

b. Tissue culture test


Laboratory Diagnosis Virulence tests - Invivo tests Bacterial growth from Loeffler’s serum slope is emulsified in 2-4 ml

broth. Two guinea pigs (GP A and GP B)

I. 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

II. 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



Virulence tests - Invitro tests

I. Elek's gel precipitation test 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



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


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


Prophylaxis

1) Active Immunization (Vaccination)

i. Formol toxoid (fluid toxoid) 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)

ii. Adsorbed toxoid (more immunogenic than fluid toxoid) purified toxoid adsorbed onto insoluble aluminium

phosphate or aluminium hydroxide given IM (DTP or TD)


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)

Schedulei) 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


Prophylaxis2. Passive immunization

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

3. Combined immunizationFirst dose of adsorbed toxoid + ADS, to be continued by the full course of active immunisation


CONTROL

1. isolate patients

2. treat with antibiotics actively

3. complete vaccination schedule should be used with booster every 5 years


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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Corynebacteria II MBBS Dr Ekta Chourasia Microbiology