C. diptheria class
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Transcript of C. diptheria class
CLASS
Corynebacterium•Diptheria
• Aerobic gram-positive bacillus
Corynebacterium
Classification Corynebacterium diphtheriae and diphtheroids
(look like C. diphtheriae) are Gram- positive, club shaped rods.
Some are saprophytic Some produce disease in animals. C. diphtheriae is the most important pathogen in the
group.
Corynebacteria
ØGram + bacillius: korynee=club shaped bacteria; diphtheria=leather hide looking pharyngeal membrane
Aerobic or facultatively anaerobic
Nonsporing ,noncapsulated &nonmotile.
Morphology
Small, pleomorphic (club-shaped rods), gram-positive bacilli that appear in short chains (“V” or “Y” configurations) or in clumps resembling “Chinese letters (cuneiform) or palisade arrangement” . This is due to incomplete separation of daughter cells after binary fission.
Øgranules composed of polymetaphosphate are seen in cells.They are more strongly gm+ve than the rest of
bacterial cell Stained with methylene blue stain granules
take up a bluish purple colour (metachromatic granules ,Babes Ernest or volutin granules).
Are often seen at poles of bacilli—(polar bodies)
Special stains- Albert, Neisser, Ponder.
Diphtheroids Gram stain
Arrangement of C. diphtheria
Corynebacterium
cultural characteristics Enrichment with blood, serum or egg ---
Incubation -35-370 C for 24 hours. pH of 7.8-8.0 for good growth.
Aerobe & facultative anaerobe -Growth on B.A – raised, translucent, gray colonies
10
Stained Corynebacterium cells. The "barred" appearance is due to the presence of polyphosphate inclusions called metachromatic granules. Note also the characteristic "Chinese-letter" arrangement of cells.
C. diphtheriae
Stained Corynebacterium cells. The "barred" appearance is due to the presence of polyphosphate inclusions called metachromatic granules. Note also the characteristic "Chinese-letter" arrangement of cells.
C. diphtheriae
• cultural characteristics cont
• Media• Selective & differential media• LOEFFLER serum slope
• Cystine-tellurite blood agar• Modified Tinsdale agar
– .
• LOEFFLER serum slope• Loeffler‘s agar slant contains serum and egg that
enhance the formation of metachromatic granules
• On this media diptheria bacilli grow very rapidly & colonies appear in 6-8 hrs.
• Colonies are at first small circular white discs & enlarge on further incubation.
Methylene blue stain from Loefflers slant
• Tellurite blood agar• Tellurite (o.o4%) inhibits growth of most bacteria,
acting as a selective agent.• Diptheria bacilli reduces tellurite to metallic
tellurium which is incorporated in the colonies giving them a black or gray color.
• Growth is delayed & colonies may take 2 days to appear.
Corynebacterium
– . Two kinds of media are used:• Cystine tellurite has a longer shelf life• Tinsdale helps to differentiate amongst the
Corynebacterium. –Colonies on either appear black or gray due
to tellurite reduction. –S. aureus and Listeria also grow as black
colonies. –On Tinsdale C. diphtheriae, ulcerans, and
pseudotuberculosis form brown halos around the colonies due to formation of ferric sulfide.
Gram +ve Bacilli and Colonies
Corynebacterium
– 3 morphological types of C. diphtheriae are found on tellurite containing media: • Gravis – large, gray colonies• Intermedius – small, dull gray to black. • Mitis – black colonies with a gray
periphery• All produce an immunologically identical
toxin.
Diphtheria Epidemiology
• Reservoir asymptomatic human carriers(nose & throat) outnumber cases
• Transmission Respiratory, aerosols Skin lesions
• Temporal pattern Winter and spring
• Communicability Upto several weekswithout antibiotics
Diptheria: Corynebacterium diptheriae
• Epidemiology: humans are the only known reservoirs– Spread is via person-person contact thru
respiratory droplets or by direct contact w/skin lesion exudates
– Usually seen unimmunized or under immunized adults in urban and poor rural areas
Virulence factors - C. diphtheriae • Toxin production occurs only
when C. diphtheriae infected by virus (phage) carrying tox gene.
• Dip. Toxin is a protein.• It is very potent (lethal dose for a 250g guinea pig is
0.0001mg.• It has 2 fragments- A (active) & B (binding).• Ab to frag B is protective by preventing binding of
toxin to cells• If isolated, must be distinguished from normal
diphtheroid• Toxoid developed in 1920s
Corynebacterium• Virulence factors C. diphtheriae
– For C. diphtherias to cause diphtheria an exotoxin must be produced.• Is a heat-labile polypeptide produced during
lysogeny of a phage that carries the "tox” gene.
• Alkaline pH of 7.8- 8.0, aerobic conditions, and a low environmental iron level are essential for toxin production (occurs late in the growth of the organism).
• The toxin inhibits protein synthesis by ADP-ribosylating elongation factor 2.
Corynebacterium(exotoxin)
• Trypsin cleaves the toxin into 2 fragments, A and B, that are linked together by a disulfide bridge.
• Fragment B is required for toxin binding to tissue cells and fragment A contains the toxic activity.
• One molecule of toxin can inhibit 90% of the protein synthesis in a cell.
• Systemic effects include heart failure, paralysis and adrenal hypofunction leading to an Addison’s like disease.
C. diphtheria toxin
• Toxin enters through receptor mediated endocytosis
• Acidification of endocytic vesicle allows A to dissociate from B
• A enters cytoplasm
Mechanism of Action of Diphtheria Toxin: Inhibition of Protein Synthesis
Corynebacterium
• Clinical Significance (C. diphtheria) – Is normally found in the throats of healthy
carriers. –The organism infects only man and it has a limited
capacity to invade.
–Diphtheria - Disease usually starts as a local infection of the mucous membranes causing a membranous pharyngitis–Local toxin effects result in degeneration of
epithelial cells. – Inflammation, edema, and production of a
pseudomembrane composed of fibrin clots, leukocytes, and dead epithelial cells and microorganisms occurs in the throat.
Corynebacterium
–The more dangerous effects occur when the toxin becomes systemic and attacks the heart(heart failure), peripheral nerves (paralysis), and the adrenal glands (hypofunction).
–Cutaneous diphtheria
– More common in tropical and subtropical areas.
–Necrotic lesions with occasional formation of a local pseudomembrane occur.
Diphtheria Clinical Features
Incubation period 2-5 days (range, 1-10 days)
May involve any mucous membraneClassified based on site of infectionanterior nasalpharyngeal and tonsillarlaryngealcutaneousoculargenital
Pharyngeal and Tonsillar Diphtheria
• Insidious onset of exudative pharyngitis
• Exudate spreads within 2-3 days and may form adherent pseudo membrane
• Membrane may cause respiratory obstruction
• Fever usually not high but patient appears toxic
• Malignant or hypertoxic dipt
• Severe toxemia, adenitis (bull neck)
Diphtheria Complications
• Mostly attributable to toxin• Severity generally related to extent
of local disease• Most common complications are
myocarditis and toxic neuritis with palsy
• Death occurs in 5%-10% for respiratory disease
Diphtheria: Corynebacterium diphtheria
• Complications:–Airway obstruction from
edema/membrane formation–CHF–Cardiac conduction
disturbances–muscle paralysis
Diphtheria - pseudomembrane
–This may obstruct the airway and result in suffocation.
Thick Membrane
Pseudo membrane
Lab Dx
• 1. Isolation of Dipt bacillus
• 2.Demonstration of toxicity/ Virulence tests
Lab Dx (cont)• Isolation of Dipt bacillus• From throat or nasopharyngeal swabs, cutaneous
swabs.• Smears made & gm staining done.• In throat swabs– have to be differentiated from
commensal corynebacteria.• Dipt bacilli may not be always be seen in smears from
lesions.• Toxigenic dipt bacilli can be identified in smears by IF.
• CULTURE
Virulence tests–To prove that an isolate can cause diphtheria, one must
demonstrate toxin production.– In vitro test (Elek gel ppt test)
–This is most often done on an Elek plate:–A filter strip containing anti-toxin antibody is placed on
the surface o the filter paper of 20% horse serum agar in a petridish perpendicular to the streak of the organism.
–Narrow streaks of strains are made at rt angles to the filter paper strip .
–A +ve & -ve control is put up.–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.
Elek plate
Virulence tests
–In vivo tests–Guinea pig
– tissue culture toxicity assays may also be done.
In vivo test forToxicity(virulence)
Corynebacterium
• treatment–Penicillin to eliminate the organism.–Erythromycin – carriers.
–Prevention-– Active immunization with toxoid (alum
precipitate). – Is part of the DPT vaccine.
–Schick skin test – it tests for circulating antibody to the toxin by
injecting a small amount of toxin intradermally and observing for a local erythematous and necrotic reaction. – If this occurs it indicates that the person has no anti-
toxin antibodies and is, therefore, susceptible to diphtheria.
Diphtheria Toxoid
• Formalin-inactivated diphtheria toxin
• Schedule Three or four doses + booster Booster every 10 years
• Efficacy Approximately 95%
• Duration Approximately 10 years
• Should be administered with tetanus toxoid as DTaP, DT, Td, or Tdap
DosePrimary 1Primary 2Primary 3Primary 4
Age2 months4 months6 months
15-18 months
Routine DTaP Primary Vaccination Schedule
4-6 yrs11-12 yrsEvery 10 yrs
DTaP, DT, and Td
DTaP, DT
Td, Tdap (adult)
Diphtheria7-8 Lf units
2-2.5 Lf units
Tetanus5-12.5 Lf units
5 Lf units
Passive immunisation( Diphtheria Antitoxin)
• Produced in horses• First used in the U.S. in 1891• Used only for treatment of
diphtheria• Neutralizes only unbound toxin
Diptheroids.
• Other Corynebacterium are part of the normal flora of the skin and URT.
• Are called diphtheroids and may occasionally cause disease, particularly in immunocompromised individuals.
Other pathogenic Corynebacteria
–C. ulcerans toxigenic strains may produce a disease similar to, but less severe than diphtheria.
–J-K Group commonly cause infections in those with underlying disease. – Diseases include bacteremia, meningitis, peritonitis,
wound infections, etc. – It is becoming more and more of a problem.
–C. pseudotuberculosis found in those with exposure to animals. – Can cause pneumonia or lymphadenitis. – Produces a different exotoxin than C. diphtheriae.
Pathogenic Corynebacterial Species
Corynebacterium diphtheriae
Corynebacterium jeikeium
Corynebacterium urealyticum
Corynebacterium urealyticum
ØUrinary tract infections (UTI’s); rare but important
ØUrease hydrolyzes urea; release of NH4+, increase
in pH, alkaline urine, renal stones
Corynebacterium jeikeium
ØOpportunistic infections in immunocompromised (e.g., patients with blood disorders, bone marrow transplants, intravenous catheters)
ØMultiple antibiotic resistance common (MDR)
ØCarriage on skin of up to 40% of hospitalized patients (e.g., marrow t-plants)
Virulence Factors in Corynebacterium Species