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