Coryneform bacteria :
Gram-positive, non-acid fast, non-motile
Irregular stained with granules
Club shaped swelling at one or both ends so the name
Important pathogen
Diptheros meaning leather
Corynebacterium diphtheriae :
Caused diphtheria
slender rods
clubbing at both ends
pleomorphic
non-capsulate/ acid-fast, gram-positive
granules are composed of polymetaphosphate
staining with Loeffler’s methylene blue show bluish-purple metachromatic granules with polar bodies
CLASSIFICATION :
Corynebacterium diphtheria and diphtheroid (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
Morphology and cultural characteristics :
small gram-positive Bacilli; arrangement = palisade or Chinese letters
growth on blood agar – raised, translucent, grey colonies
Classification McLeod :
Classified in 3 types
gravis : produce most serious hemorrhagic; paralytic complications- epidemic
intermedius : hemorrhagic
mitis: obstructive complications, endemic geographic locations differ, testing for toxigenic is more important
Corynebacterium :
3 morphological types of C.diphtheria are found on tellurite containing media :
Mitis: black colonies with a grey periphery
Gravis: large, grey colonies
Intermedius: small, dull grey to black
all produce an immunologically identical toxin
CULTURAL CHARCTERISTICS :
Incubation 35-37⁰C for 24 hours
they prefer a pH of 7.8 – 8.0 for good growth
they require access to oxygen
Catalase positive
RESISTANCE :
can be killed at 58⁰C in 10 minutes
100⁰C in 1 minute
survive in blankets, floor dust, toys inanimate objects
What is Diphtheria …?
An infection of local tissue of upper respiratory tract with the production of toxin which causes systemic effects on the heart and peripheral tissues.
Pharyngeal diphtheria :
Insidious onset of exudative pharyngitis
Exudates spread to form adherent “pseudomembrane”
can lead to respiratory obstruction and death by asphyxiation
Fever not high, but the patient appears toxic
Staining methods :
Gram’s method
Albert’s stain
Neisser’s stain
Pander’s stain
TOXIN :
Pathogenicity associated with toxin
Gravis/ intermedius 95-99% are toxigenic
Miltis 80-85%
Some abundant others poorly
Toxin production park William 8
Toxin: M.W. 62,000
Toxin contains 2 components: A & B
A produce toxigenicity by the proteolytic effect
B produces binding
Toxin + Formalin = Toxoid (antigenic, not toxigenic)
Toxin inhibits protein synthesis
Fragment A catalyzes the transfer of ADP ribose from the Nicotinamide adenine dinucleotide to the eukaryotic elongation factor 2
Causes involvement with affinity.
Myocarditis
Adrenals
Nerve endings
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
Diphtheria- Pseudo-membrane : this may obstruct the airway and result in suffocation.
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.
Animal testing (obsolete)
PATHOGENICITY :
Bacteria invade, colonise, proliferate
Bacteria are lysogenised by beta phage
Produce toxin
Kills epithelial and neutrophils
Produce pharyngitis and cutaneous lesions
Incubation 3 – 4 days/ one day
Faucal/ Nasal/ Laryngeal/ Otic/ Conjuctival/ Genital/ Vulvae and cutaneous manifestations
Diphtheria is a toxemic condition
Malignant sever toxaemia, Adenitis Bull. Neck circulatory failure, septic gangrene, pseudo membrane, haemorrhagic Epistaxis, purpura, general bleeding tendency, Asphyxia, acute circulatory failure, paralysis pneumonia, septic shock, otitis media, toxaemia, necrotic changes, death in guinea pigs.
C. diphtheria :
C. diphtheria occurs in the respiratory tract, in wounds, or on the skin of infected preson or normal carriers.
It is spread by droplets or by direct contact
Portal of entry : respiratory tract or skin abrasions.
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.
TOXICITY AND DIPHTHERIA TOXIN :
Local toxigenic effects : elicit inflammatory responses and necrosis of the faucial mucosa cells formation of pseudo membrane (composed of bacteria, lymphocytes, plasma cells, fibrin, and dead cells), causing respiratory obstruction.
Systemic toxigenic effects: necrosis in heart muscle, liver, kidneys and adrenals. Also produces neural damage.
COMPLICATIONS :
Asphyxia – causing mechanical obstruction
May need tracheotomy
Circulatory failure
Post diphtheria paralysis
NON TOXIGENIC CLINICAL MANIFESTATIONS :
Bacteria can produce :
Endocarditis
Meingitis
Cerebral abscess
Osteoarthritis
DIAGNOSIS OF DIPHTHERIA :
A) LABORATORY DIAGNOSIS :
Specific treatment is more important than Laboratory diagnosis : Isolation of diphtheria bacilli; testing for toxigenicity
B) COLLECTION OF SPECIMENS :
Throat swabs; smear examinations ( Gram’s staining; albert’s & Ponder’s staining); immuno-fluroscent methods; culture on Loeffer’s serum slope & blood agar.
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
In Vitro : 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.
Schick test : injection of toxin intradermal route
Produces redness/ erythematic in 2-4 days
No reaction – protective immunity present
EPIDEMIOLOGY :
Eradicated in developed nations
Children between 2-5 years
A systematic carriers
Person to person contact
Carriers spread
Prolonged contact
PROPHYLAXIS :
Immunization
Active – Passive
Both passive and Active
Herd Immunity
Schick test
Immunization with Antitoxin
