Mircrobiology Lecture -16 Diptheria

Diphtheria

Diphtheria

Taxonomy

Genus: Corynebacterium Species: C.diphtheriae

Diphtheria

Morphology Gram positive rod clubbing at both ends metachromatic granules (volutin). nonsporing, noncapsulated and nonmotile appear in pairs, palisades, or small group (Chinese letters).

Diphtheria

Cultural characteristics Facultative anaerobe; Requires nutrional rich media, such as Loeffler’s serum and tellurite blood agar; Most strains grow well on lipid-free media; Types of colonies: mitis, garvis and intermedius;

Corynebacterium diphtheriae colonies on blood agar Diphtheria

Cultural characteristics

Corynebacterium diphtheriae hem+

Corynebacterium diphtheriae hemDiphtheria

Cultural characteristics

C.Diphtheriae gravis

C.Diphtheriae mitis

Diphtheria

Morphological and cultural properties of Corynebacterium diphtheriae Gravis

Intermedius

Mitis

Morphology

Short rods, few or no valutine granules

Long, curved, Forms with clubbed pleomorphic ends, poor granulation, rods with very pleomorphic prominent

Colony on tellurite blood agar

In 18 hours, colony is 1-2 mm in size, with greyish black centre, paler, semitranslucent periphery

18 hour colony small, 1mm in size, misty

Consistency of colonies

Intermediate Moves as a whole on the Soft, buttery, plate, not easily picked out easily or emulsifiable Between gravis ant mitis emulsifiable

Hemolysis

Variable

Nonhemolytic

Shiny black

Usually hemolytic Diphtheria

Corynebacterium diphtheriae: tests to identify and distinguish gravis, intermedius and mitis biotypes

Organism

Sucrose Mannitol Starch Glucogen hydrolysis

Nitrate Urease produc tion

D

toxin

C.diphtheriae var.gravis

+

+

+

+

+

+

C.diphtheriae var. intermedius

-

+

-

-

+

+

C.Diphtheriae var.mitis

+

+

-

-

+

+

C.ulcerans

-

-

+

+

+

Cp

+

Both toxins

+

Diphtheria

Epidemiology Worldwide distribution maintained in asymptomatic carriers and unvaccinated hosts; Humans are the only known reservoir. With carriage in oropharynx or on skin surface; Spread person to person by exposure to respiratory droplets or skin contact; Disease observed in unvaccinated people, children or adults with waning immunity;

Diphtheria

Respiratory diphtheria

Diphtheria

Cutaneous diphtheria

Diphtheria

Virulence factors

Adhesive factors- pili, microcapsule? Invasive factors- hyaluronidase, neuraminidase; A-B exotoxin that inhibits protein synthesis by inactivating elongation factor (EF-2);

Diphtheria

The Beta phage that encodes the tox gene for the diphtheria toxin

Strains of Corynebacterium diphtheria which elaborate exotoxin (tox+) are lysogenic (infected with a specific bacteriophage-β).

Diphtheria

Toxin  Exotoxin is a heat-labile polypeptide;  Consist of 2 fragments: A and B;  When released by the bacterium the toxin is inactive because the active site on fragment A is masked;  Activation is probably is accomplished by proteases group in culture medium and infected tissues;  All the enzymatic activity of the toxin present in fragment A;  Fragment B is responsible for binding the toxin to the cells. The receptor for the toxin is the heparin-binding epidermal growth factor (present on the surface of many eukaryotic cells);

Diphtheria

Toxin  The diphthery toxin is a cytotoxin;  It’s mechanism of action is inhibition of protein synthesis;  Inhibition of protein synthesis is responsible for both the necrotic and neurotoxic effect of the toxin;  The antibody to fragment B is protective by preventing the binding of the toxin to the cell;  Prolonged storage, incubation at 37 C for 4 to 6 weeks , treatment with 0.2 to 0.4 % formalin or acid pH converts it to toxoid;  Toxoid is toxin that has lost its toxicity but not its antigenicity which is capable of inducing antitoxin and reacting specifically with it; Diphtheria

Toxin The Diphtheria Toxin (DTx) Monomer. A (red) is the catalytic domain; B (yellow) is the binding domain which displays the receptor for cell attachment; T (blue) is the hydrophobic domain responsible for insertion into the endosome membrane to secure the release of A. The protein is illustrated in its "closed" configuration. Diphtheria

The Mechanism of action of Diphtheria toxin DTxA

Diphtheria

Mechanism

EF-2+ NAD+→ADP-RIBOSE-EF-2+ NICOTINAMIDE + H+

Diphtheria toxin catalyzes the ADP-ribosylation, and inactivates, the elongation factor eEF-2. In this way, it acts to inhibit translation during eukaryotic protein synthesis

Diphtheria

Pathogenesis of Diphtheria Diphtheria is an upper respiratory tract illness characterized by sore throat, low fever, and an adherent membrane (called a pseudomembrane) on the tonsils, pharynx, and/or nasal cavity. Diphtheria toxin produced by C. diphtheriae, can cause myocarditis, polyneuritis, and other systemic toxic effects. A milder form of diphtheria can be restricted to the skin.

Diphtheria

The clinical appearance of Diphtheria

Diphtheria: the off white, smooth pseudomembrane affects the tonsils and pharynx.

Diphtheria

Diphtheria: bull-neck appearance and tracheostomy

Diphtheria

Treatment  Obstruction of throat with pseudomembranes may require intubation or tracheotomy.  Early use of diphtheria antitoxin is necessary for neutralization of exotoxin.  Antitoxin is derived from horses, so test for hypersensitivity is needed.  Use of penicillin or erythromycin to eliminate C.diphtheriae and terminate toxin production.

Diphtheria

Prevention and Control  Administration of diphtheria vaccine (toxoid) during childhood and booster doses every 10 years through life.  Toxoid is given together with tetanus toxoid and pertussis vaccine (DTP).  Toxoid is prepared by formalin treatment of the diphtheria toxin.

Diphtheria

Laboratory diagnosis Microscopy is nonspecific. Special stains Neisser’s or Loeffler’s have been devised for demonstrating the volutin granules.  Culture is perfomed on selective media (Tinsdale’s agar, which contain potassium tellurite).  Toxigenicity testing. Demonstration of exotoxin is performed by molecular or immunologic methods (immunodiffusion assay).

Diphtheria

Corynebacterium Species Associated with human Disease Organism

Disease

C.diphtheriae

Diphtheria (Respiratory, cutaneous); Pharyngitis and endocarditis (nontoxigenic strains)

C.jeikeium (group JK)

Septicemia, endocarditis, wound infections, foreign body (catheter, shunt, prosthesis) infections

C.urealyticum (group D2)

Urinary tract infections including pyelonephritis and alkaline-encrusted cystitis, septicemia, endocarditis, wound infections.

C.amycolatum

Wound infections, foreign body infections, septicemia, urinary tract infections, respiratory tract infections

C.macginleyi

Eye infections

Lymphadenitis, ulcerative lymphangitis, abscess C.pseudotuberculosis formation C.ulcerans

Respiratory diphtheria Diphtheria

Summary of Other Corynebacterium species Physiology and structure  Gram-positive bacilli with an irregular shape;  Some species reguire lipids for good growth (e.g.,C.jeikeium, C.urealyticum, C.macginleyi);  Most strains are facultative anaerobes;

Diphtheria

Virulence  A-B exotoxin may be carried by C.ulcerans and C.pseudotuberculosis;  Urinary tract pathogens produce urease (e.g.,C.amycolatum, C.glucuronolyticum, C.riegelli, C.urealyticum);  Many species able to adhere to foreign bodies (e.g.,catheters, shunts, prosthetic devices);  Some species resistant to most antibiotics (e.g., C.amycolatum, C.jeikeium, C.urealyticum);

Diphtheria

Epidemiology Most infections are endogenous (produced by species that are part of the host’s normal bacterial population on the skin surface and mucosal membranes).

Diphtheria

Epidemiology of Corynebacterium Organism

Habitat (reservoir)

Mode of Transmission

Corynebacterium diphtheriae

Inhabits human nasopharynx but only in carrier state, not considered part of normal flora isolation from healthy humans is not common

Corynebacterium jeikeium

Uncertain, may be person to person Skin flora of hospitalized or selection of endogenous patients, most commonly resistant strains during in the inguinal, axillary, antimicrobial therapy, introduced and rectal sites during placement or improper care of intravenous catheters

Person to person by exposure to contaminated respiratory droplets or direct contact with infected cutaneous lesions, may also be transferred by exposure to contaminated objects.

Diphtheria

Epidemiology of Corynebacterium Organism Corynebacterium ulcerans

Habitat (reservoir)

Mode of Transmission

Uncertain, asotiated with close Normal flora of humans animal contact, especially during and cattle. summer

Assotiated with infections in animals Corynebacterium pseudotuberculosis such as sheep, goats, and horses

Uncertain, assotiated with close animal contact, but infections in humans are rare

Corynebacterium urealyticum

Normal human skin flora

Uncertain, probably by access of patient’s endogenous strain to normally sterile site

Corynebacterium xerosis

Normal flora of human conjunctiva, skin, and nasopharynx

Uncertain, probably by access of patient’s endogenous strain to normally sterile site Diphtheria

iseases epticemia, endocarditis, foreign body infections, wound infections, urinary tract infections, respiratory infections including diphtheria. iagnosis ulture on selective and nonselective media. Growth may be slow and media may require supplementation with lipids. reatment, Prevention, and Control Diphtheria

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