Diphtheria, Corynebacterium diphtheria

Diphtheria Corynebacterium diphtheria

Kleb’s – Loffler's bacilli.

Dr.T.V.Rao MD

Dr.T.V.Rao MD 1

Corynebacterium

• Gram + Non Acid fast, Non motile,

• Irregularly stained with granules,

• Club shaped swelling at one or both ends

so the name

• Important Pathogen

Corynebacterium diphtheria,

Diptheros meaning leather,

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Corynebacterium diphtheria

• Slender rods

• Clubbing at both ends

• Pleomorphic

• Non capsulate / Acid fast Gram +

• Granules are composed of polymetapohosphate

• Staining with Loeffler’s methylene blue show bluish purple metachromatic granules. with polar bodies,

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Corynebacterium diphtheria

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Corynebacterium

• Classification – Corynebacterium diphtheria and

diphtheroids (look like C. diphtheria) are Gram- positive, club shaped rods.

• Some are saprophytic

• Some produce disease in animals.

• C. diphtheria is the most important pathogen in the group.

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Corynebacterium

• Morphology and cultural characteristics

–Small Gram positive Bacilli; arrangement=palisade or Chinese letters

–Growth on Blood agar – raised, translucent, gray colonies

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Classification McLeod

Classified in to 3 Types 1 Gravis

2 Intermedius

3.Mitis Gravis produce Most serious Hemorrhagic

Paralytic complications - Epidemic

Intermedius Hemorrhagic

Mitis - obstructive complications, Endemic

Geographic locations differ

Testing for toxigenicity is more important, Dr.T.V.Rao MD 7

Corynebacterium

–3 morphological types of C. diphtheria are found on tellurite containing media:

• Mitis – black colonies with a gray periphery

• Gravis – large, gray colonies

• Intermedius – small, dull gray to black.

• All produce an immunologically identical toxin.

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Antigenic structure

• Gravis 13,

• Intermedius 4

• Mitis 40

• Bacteriophage typing 15 types

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Cultural Characteristics

–Incubation -35-370 C for 24 hours.

–They prefer a pH of 7.8-8.0 for good growth.

–They require access to oxygen (poor AnO2 growth).

–Catalase +

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Resistance

• Can be killed at

58o c in 10 minute

100oc in 1 minute

Survive in Blankets, Floor dust,

toys inanimate objects

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What is Diphtheria • An infection of

local tissue of

Upper respiratory

tract with

production of toxin

which causes

systemic effects on

Heart and

Peripheral tissues,

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Pharyngeal diphtheria

• Insidious onset of exudative

pharyngitis

• Exudate spreads to form adherent

“pseudo membrane”

– can lead to respiratory obstruction

and death by asphyxiation

• Fever not high, but patient appears

toxic Dr.T.V.Rao MD 13

Staining methods

• Gram’s method

• Albert's stain

• Neissers stain

• Pander's stain

• On staining seen as

Pairs, Appear as v and L letters, resembling

Chinese letter pattern or also called

cuneiform arrangement. Dr.T.V.Rao MD 14

Cultural characters

• Need enrichment Media

• Containing

• Blood, Serum or Egg 370 c ph. 7.4

• Aerobic/Facultative anaerobic.

• Commonly used medium

• Loeffler serum slope,

• Tellurite Blood agar,

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Cultural characters

• Loeffler serum slope

Grows rapidly in 6 -8 hours,

Small white opaque disks

Turns to yellow

Tellurite blood agar Modified Mc Leod and Hoyle’s Medium

Commonly used medium

Tellurite blood agar Contains tellurite 0.04 tellurite Inhibits other bacteria

Produce Grey/Black colonies.

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Biochemical Reactions • Acid production on

Glucose,Galactose Maltose, Dextrin

Do not produce acid with

Lactose, Mannitol, sucrose.

All fermentation reactions tested in

Hiss serum sugars

Urease test negative.

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Toxin • Pathogenicity associated with Toxin

• Gravis/Intermedius 95-99% are toxigenic

• Mitis 80 – 85%

• Some abundant others poorly

• Toxin production park William 8

• Toxin

M W 62,000

0.0001mg can kill guinea pig weighing 250 grams

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C. diphtheria toxin

• Toxin enters

through receptor

mediated

endocytosis

• Acidification of

endocytic vesicle

allows A to

dissociate from B

• A enters

cycoplasm Dr.T.V.Rao MD 19

Toxin ( Contd ) • Toxin contain two components

A 24,000

B 38,000

A produce toxigenicity by proteolytic effect

B Produce binding

Toxin + Formalin = Toxoid

What is Toxoid – Antigenic, not toxigenic

Tox + Corynephage Toxin production

Dr.T.V.Rao MD 20

Toxin ( contd ) • Need iron 0.1 mg/liter.

• Toxin inhibits protein synthesis

• Fragment A catalyzes the transfer of ADP ribose from the Nicotinamide adenine dinucleotide ( NAD ) to the eukaryotic elongation factor 2 /(Fragment A inhibits polypeptide chain elongation in the presence of Nicotinamide adenine dinucleotide by inactivating elongation factor

• Causes involvement with affinity.

Myocarditis,

Adrenals

Nerve endings,

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Clinical features

• Malaise, Sore throat, Fever

• Adherent grey pseudo membrane

• Nasal ulcers,

• Obstruction of larynx and lower airways,

• Difficulty in swallowing

• Lead to Myocarditis, Peripheral neuritis,

• Paralysis of limbs,

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Diphtheria - Pseudo

membrane • This may obstruct the airway and result in

suffocation.

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Corynebacterium

–To prove that an isolate can cause diphtheria, one must demonstrate toxin production. • This is most often done on an Elek plate:

– The organism is streaked on a plate containing low iron.

– A filter strip containing anti-toxin antibody is placed perpendicular to the streak of the organism.

– Diffusion of the antibody into the medium and secretion of the toxin into the medium occur.

– At the zone of equivalence, a precipitate will form.

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Elek plate for Demonstration

of Toxin

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Animal testings (obsolete)

Pathogenicity

• Bacteria Invade, Colonise,Proliferate

• Bacteria are lysogenized by Beta

phage

• Produce toxin,

• Kills epithelial and Neutrophils,

• Produce Pharyngitis and cutaneous

lesions.

Dr.T.V.Rao MD 27

Dr.T.V.Rao MD 28

Pathogenicity

• Incubation 3 – 4 days / one day

• Faucal / Nasal /Laryngeal / Otic /

Conjunctival,/Genital / Vulvae

and Cutaneous Manifestations

Diphtheria is a toxemic condition.

Malignant Sever toxemia ,Adenitis Bull

neck Circulatory failure

Septic Gangrene , pseudo membrane.

Dr.T.V.Rao MD 29

Pathogenicity • Hemorrhagic

Epistaxis ,

Purpura

General Bleeding tendency

Asphyxia , Acute circulatory failure,

Paralysis Pneumonia, Septic shock, Otitis media. Toxemia, Necrotic changes

Death in Guinea pigs

Dr.T.V.Rao MD 30

C. diphtheria Pathogenesis and Immunity

C. diphtheria occurs in the

respiratory tract, in wounds, or on

the skin of infected persons or

normal carriers. It is spread by

droplets or by direct contact.

Portal of entry: respiratory tract or

skin abrasions. Dr.T.V.Rao MD 31

Diphtheria bacilli produce Toxin

• Diphtheria bacilli

colonize and grow on

mucous membranes,

and start to produce

toxin, which is then

absorbed into the

mucous membranes,

and even spread by

the bloodstream.

Dr.T.V.Rao MD 32

Toxicity of Diphtheria Toxin

• Local toxigenic effects: elicit

inflammatory response and

necrosis of the faucial mucosa

cells-- formation of "pseudo-

membrane“ (composed of

bacteria, lymphocytes, plasma

cells, fibrin, and dead cells),

causing respiratory obstruction.

Dr.T.V.Rao MD 33

Systemic Toxigenic effects

• Systemic

toxigenic effects:

necrosis in heart

muscle, liver,

kidneys and

adrenals. Also

produces neural

damage.

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Complications

• Asphyxia - causing

mechanical obstruction.

• May need tracheotomy

• Circulatory failure.

• Post Diphtheria paralysis

Dr.T.V.Rao MD 36

Non toxigenic clinical

manifestations

• Bacteria can produce

1Endocarditis,

2Meingitis,

3 Cerebral abscess.

4 Osteoarthritis.

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Diagnosis of Diphtheria

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Laboratory Diagnosis

• Specific treatment is more

important than Laboratory

Diagnosis.

1 Isolation of Diphtheria bacilli.

2.Testing for toxigenicity,

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Collection of Specimens

• Throat swabs

• Smear examinations

Gram s staining,

Alberts, Ponders

Immunoflorescent methods

Cultures on Loeffers serum slope

Tellurite Blood agar,

Blood agar.

Dr.T.V.Rao MD 40

Dr.T.V.Rao MD 41

Growing the C.diptheria

• Serum slope – Growth in 6 – 8 hours,

• Stain with Neisse's stain, Albert's

stain

• Bacilli have metachromatic granules,

• Tellurite Blood agar takes two days

for manifestation of colonies,

Dr.T.V.Rao MD 42

Colonial morphology

Black colonies on tellurite agar

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Virulence tests, • In Vivo and In Vitro

• In Vivo in Animals

• Subcutaneous tests

Inject broth from culture into two Guinea pigs, 0.8 ml

One animal given 500 units of antitoxin before

Other no Antitoxin.

Animal not given antitoxin will die

Leads to Loss of Animals. So we do not do it at present

Dr.T.V.Rao MD 44

In Vitro Testing

• Elek’s Gel precipitation testing

• Filter paper impregnated with Diphtheria

antitoxin 1000 Units / ml

• Tested on the horse serum agar

• Positive / Negative /Test strains tested for

Immunodiffusion

• Line of precipitation – test positive

• Other methods testing in Tissue cultures.

Dr.T.V.Rao MD 45

Elek’s Test for In vitro

toxigenicity Testing

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Schick Test

• Injection of toxin

Intradermal route

• Produces

redness/erythemati

c in 2-4 days

• No reaction –

Protective

immunity present.

Dr.T.V.Rao MD 47

Epidemiology

• Eradicated in developed nations,

• Children between 2 – 5 years.

• A symptomatic carriers

• Person to person contact.

• Carriers spread.

• Prolonged contact.

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Prophylaxis • Immunization

• Active – Passive

• Both passive and Active.

• Herd Immunity.

• Schick test

• Immunization with Antitoxin

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Active Immunization.

• Toxoid – Toxin treated with Formaldehyde

• Absorbed Toxoid

• Given by Intramuscular route

• Given in DTP –Triple Vaccine

• Primary Immunization

• Three Doses of DPT at least 4 weeks apart.

• Non vaccinated

• Three doses of Toxoid four weeks apart

• One dose after One Year.

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Passive Immunization

• Given in Acute infections

• Give Subcutaneously

• 500 – 1000 Units of Antitoxin

• Given as Horse Serum

• Combined in Acute Infections (

Both Active Immunization with

Toxoid and Antitoxin.

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Treatment • Antibiotic not useful in Acute infections,

• Antitoxin a must and primary concern

• Anti toxin obtained from horse serum

• Mild 20,000 to 40,000

• Moderate 40,000 to 60,000

• Severe 80,000 to 1,00,000

• Commonly used antibiotics,

• Penicillin parentally,

• Oral Erythromycin

Dr.T.V.Rao MD 52

Treating Contacts

•All contacts are

advised to receive

500 mg

Erythromycin 4 times

a day. Dr.T.V.Rao MD 53

Summary • Diphtheria is still prevalent in many

parts of the world

– Risk of imported disease and outbreaks

– Continuing need for vaccination

• Toxin-mediated disease

• Largely unexplored pathogenesis

• Immense opportunities for research, especially in the post-genomic era

Other Corynebacterium

• C.ulcerans

• Like C.diptheria

• Like Gravis type gelatin liquefied

• Transmitted through cows Milk

• Erythromycin effective.

• Diphtheria antitoxin is protective.

Dr.T.V.Rao MD 55

Diptheroids

• Resembles C.diptheria

• Commensals in throat, skin,

• C.hofmani

• C.xerosi

• Propioniebacterium

• P.acnes P.granulosum

Dr.T.V.Rao MD 56

Other Corynebacterium Species They are ubiquitous in plants and animals. Many are found as

part of human normal flora and may cause opportunistic

infections, such as pneumonia, endocarditis, and soft tissue

and bone infections, in immunocompromised patients.

C. jeikeium: sepsis, endocarditis, wound infections,

foreign body infections.

C. urealyticum causes UT infections. It is a strong urease

producer, infection of UT may lead to formation of stones.

C. ulcerans is closely related to C. diphtheriae. May cause

diphtheria-like disease.

Resistant to many antibiotics. Treatment of bacteremia or

endocarditis must be guided by antibiotic susceptibility tests. Dr.T.V.Rao MD 57

Other Corynebacterium Species

• They are ubiquitous in plants and animals.

Many are found as part of human normal

flora and may cause opportunistic

infections, such as pneumonia,

endocarditis, and soft tissue and bone

infections, in immunocompromised

patients.

• C. jeikeium: sepsis, endocarditis Dr.T.V.Rao MD 58

Other Corynebacterium Species

• C. urealyticum causes UT infections. It is a

strong urease producer, infection of UT may

lead to formation of stones.

• C. ulcerans is closely related to C.

diphtheriae. May cause diphtheria-like

disease.

• Resistant to many antibiotics. Treatment of

bacteremia or endocarditis must be guided by

antibiotic susceptibility tests. Dr.T.V.Rao MD 59

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Dr.T.V.Rao MD for Medical

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Email

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