CLIN BACTER: MOD 5 UNIT 1

• Klebs-Loeffler's bacillus


  Common name for Corynebacterium diphtheriae
• Edwin Klebs first observed the bacterium in diphtheritic membranes


  1883

  
• Friedrich Löeffler cultivated the bacterium


  1884
• Corynebacterium diphtheriae

  • Gram-positive bacilli
  • Pleomorphic, possessing irregular swellings at one end that give them the "club-shaped" appearance
  • Arranged in pairs of cells at angles to one another (X, Y V, or L configuration, or "Chinese letters"); or single cells that tend to lie parallel to each other ("palisades")
  • Metachromatic granules (called volutin granules or Babe-Ernst granules) are irregularly distributed in the cytoplasm (often near the poles) that give the rod a beaded appearance in stained smear
  • Nonmotile
  • Non-spore-forming
  • Aerobic/Facultative anaerobic
  • Catalase(+)
  • Oxidase (+)
• Habitat

  The human nasopharynx
• Transmission


  Through direct contact with respiratory secretions or exudates from skin lesions
• Diphtheria pathogenesis


  1. Incubation period of 2 to 5 days, with a range of 1 to 10 days

  2. Gradual onset of pharyngitis; early symptoms include sore throat with low-grade fever and dyspnea

  3. Pseudomembrane forms in the throat (i.e., gray-white patches composed of fibrin, necrotic host cells and bacteria) over the pharynx, tonsils, uvula, and palate

  4. Extension of membrane formation into the larynx, or anterior nasal may result in respiratory obstruction (suffocation), coma, and death

  5. Marked edema of the submandibular areas and the anterior neck along with lymphadenopathy giving a characteristic "bull neck" appearance

  6. Major complications include myocarditis and polyneuritis, and also nephritis and thrombocytopenia

  7. Paralysis of eye muscles, limbs, and the diaphragm can occur after the fifth week

  8. Damage to the heart causes heart failure, which is the most common cause of mortality in diphtheria

  9. Death occurs in 5-10%
• Cutaneous diphtheria


  • Presents as infected skin lesions which lack a characteristic appearance; a membrane forms on the infected wound that fails to heal
  • May be associated with non-toxigenic strains of C. diphtheriae
  • Appears to result less frequently in systemic complications
• Diphtheria exotoxin (Diphtherotoxin)


  • Produced by lysogenized strains of C. diphtheriae infected by β-prophages (corynebacteriophages) that carry the tox gene
  • Absorption of toxins through the mucous membranes and into the blood circulation causes local tissue destruction damage to the peripheral nervous system, heart, and other organs of the body
  • Fragment A interacts metabolically with factors in the cytoplasm and stops protein synthesis by inhibiting polypeptide chain elongation
  • Fragment B binds to and facilitates the entry of the toxin into the cytoplasm of the cells of the heart and nervous system through receptor-mediated endocytosis
• Corynebacterium ulcerans and Corynebacterium pseudotuberculosis


  • Closely related to C. diphtheriae and may carry the diphtheria tox gene
  • Whereas the toxigenic C. ulcerans can cause disease similar to clinical diphtheria, C. pseudotuberculosis rarely causes disease in humans
• Diphtheria prevention and control

  1. Diphtheria can be easily prevented by a series of vaccinations with diphtheria toxoid

  2. Persons with suspected respiratory diphtheria should be promptly given diphtheria antitoxin, produced in horses, in adequate dosage, without waiting for laboratory confirmation

  3. Diphtheria infections are also managed by chemotherapy, i.e., patients are treated with antibiotics

  4. Schick test is performed to determine susceptibility (due to lack of antitoxins), or immunity (due to presence of circulating antitoxins) of an individual against diphtheria, and hypersensitivity to diphtheria toxin or other proteins of the diphtheria cell
• Schick test

  • Involves injection of 0.1 mL of diphtheria toxin on the test arm (TA) and 0.1 mL of diphtheria toxoid (vaccine preparation or heat-inactivated toxin) on the control arm (CA)
  • The injection site (TA and CA) is inspected daily up to the 6th day for erythema, induration or necrosis which consists of a positive reaction
  • Positive reaction: TA - reaction persists until the 6th day, CA - no reaction (Susceptible; NOT hypersensitive)
  • Negative reaction: TA - no reaction, CA - no reaction (Immuned; Not hypersensitive)
  • Combined reaction: TA - reaction persists until the 6th day, CA - reaction peaks at about 48 hours and subsides by day 5 (Susceptible and hypersensitive)
  • Pseudoreaction: TA - reaction subsides by day 5, CA - reaction subsides by day 5 (Immuned and hypersensitive)

  
• Laboratory diagnosis of diphtheria

  1. Specimens: Swabs from the oropharynx, nasopharynx, or from cutaneous lesions

  2. Microscopy: Gram staining shows pleomorphic gram-positive rods that occur in angular arrangements (commonly referred to as Chinese letters or palisades) and whose ends may be swollen, producing a club shape; Loeffler's alkaline methylene blue (LAMB) staining shows pleomorphic beaded rods with reddish purple metachromatic granules or bars of polyphosphates

  3. Culture: 5% sheep BAM, Columbia CNA agar to screen and rule out group A β-hemolytic streptococci; Media containing cystine and potassium tellurite (Tinsdale agar, Cystine-tellurite blood agar) are selective and differential for Corynebacterium species

  
• Tellurite reductase activity


  Reduction to tellurium, resulting in black colonies
• C. diphtheriae stained with LAMB


  • Produces distinctive, club-shaped, diphtheroidal cells containing metachromatic granules
• C. ulcerans and C. pseudotuberculosis


  • Resemble C. diphtheriae, producing distinctive, club-shaped, diphtheroidal cells containing metachromatic granules

  
• Tinsdale agar


  Bovine serum and horse serum provide essential growth factors, sodium thiosulfate provides sulfur for H2S production, and cystine detects cystinase activity producing brown halos around the colonies

  
• Cystine-tellurite blood agar (CTBA)


  A heart infusion agar supplemented with 5% rabbit blood, tellurite, and L-cystine, a selective and differential medium for Corynebacterium species similar to TIN

  
• Loeffler's serum medium


  Contains eggs and beef serum, rather than agar, to coagulate to produce a solid medium, stimulates the growth of distinctive, club-shaped, diphtheroidal cells of C. diphtheriae and the production of metachromatic granules in the cells
• Pai medium


  Contains coagulated egg in distilled water and glycerin

  
• Inoculation and incubation


  Specimens should be inoculated to a blood agar and a selective medium such as a tellurite plate (eg, CTBA or TIN), incubate at either in ambient air or in 5% to 10% CO2 for 24 - 48 h
• Colonial characterization of C. diphtheriae


  • May appear as four distinct colony types (biotypes) designated gravis, mitis, intermedius, and belfanti, which also differ slightly in Gram's stain morphology, certain biochemical reactions, and historically, in the severity of the disease processes they produce
• Colonies of C. diphtheriae on BAM

  • Range from small, gray, and translucent (biotype intermedius) to medium, white, and opaque (biotypes mitis, belfanti, and gravis), C. diphtheriae biotype mitis may be β-hemolytic
• Colonies of C. diphtheriae on CTBA

  • Appear black or gray
• Biotypes of C. diphtheriae

  • Gravis
  • Intermedius
  • Mitis
  • Belfanti
• C. diphtheriae biotype gravis

  • Large (2-4 mm), flatter, dark gray with radial striations and irregular edges, "DAISY HEAD" colonies, short, coccoid, or pyriform
• C. diphtheriae biotype intermedius

  • Small (0.5 mm), sometime pinpoint, flat, and gray, "FROG'S EGGS" colonies, highly pleomorphic, from very long to very short rods
• C. diphtheriae biotype mitis

  • Medium-sized (1-2 mm), convex, very black with regular edges, "COOLIE HAT" colonies, may be weakly β-hemolytic, long, pleomorphic, rigid club-shaped rods
• C. diphtheriae biotype belfanti

  • -
• Colonies of C. diphtheriae on TIN

  • All four biotypes grow as black colonies surrounded by dark brown halos
• Other diphtheroids, S. aureus and some streptococci which can also grow in the presence of tellurite will develop black colonies due to tellurite reduction although brown halos around the colonies are NOT present
• Reverse CAMP reaction


  Based on the production by the organism of phospholipase C or D that inhibits the staphylococcal β-hemolysin, an arrowhead zone of NO hemolysis is formed at the junction of the organism being tested with the staphylococci
• C. ulcerans and C. pseudotuberculosis resemble C. diphtheriae, producing black colonies that are surrounded by brown halos on TIN
• Biochemical characteristics of Corynebacterium species

  • C. diphtheriae
  • C. ulcerans
  • C. pseudotuberculosis
  • C. jeikeium
  • C. pseudodiphtheriticum
  • C. xerosis
  • C. minutissimum
  • C. striatum
  • C. urealyticum
  • C. amycolatum
• Toxigenicity test


  Once an organism is biochemically identified as a possible C. diphtheriae, the isolate must be tested for the ability to produce diphtheria toxin using the Elek test, which demonstrates toxin production by C. diphtheriae

  
• Elek test

  • A filter paper strip impregnated with diphtheria antitoxin is buried just beneath the surface of a rabbit serum agar plate before the agar hardens, strains to be tested and known positive and negative toxigenic strains are streaked on the agar's surface in a line across the plate and at a right angle to the antitoxin paper strip, after 24 hours of incubation at 37°C, the plates are examined with transmitted light for the presence of fine precipitin lines at a 45-degree angle to the streaks, the presence of precipitin lines indicates that the strain produced toxin that reacted with the homologous antitoxin

  
• Corynebacterium jeikeium is most commonly isolated from acutely ill or immunocompromised patients, produces infections, including bacteremia that have a high mortality rate and is resistant to many commonly used antimicrobial drugs

  
• Corynebacterium pseudodiphtheriticum is a normal inhabitant of the human nasopharynx, can colonize natural and artificial heart valves, and has been associated with respiratory tract infections

  
• Corynebacterium xerosis normally lives in the eye, skin, and mucous membranes and is an occasional opportunist in eye and postoperative infections
• Corynebacterium minutissimum is the causative agent of erythrasma, a superficial skin infection characterized by small, brown-red macular areas, commonly of the axillary and inguinal skin, and also between the toes and finger, axilla, it gives a coral (brick red) fluorescence when exposed to Wood's light (long-wave UV radiation, also called black light) due to porphyrin