Corynebacterium diphtheriae Part-1
| Gram positive rods, non sporing, 3 - 6μm
| Club shaped Personal Notes
| causes Diphtheria (more common - respiratory diphtheria)
| a/k/a Klebs - Loeffler’s bacillus
| Observed by Klebs and cultivated by Loeffler’s
| Facultative anaerobe
| Fastidious (does not grow in nutrient agar)
| Non motile, non capsulated
| Catalase +ve and Oxidase -ve
| Chinese letter or cuneiform arrangement (failure of the daughter cells to separate
from parent cell → k/a Snapping type of cell division)
CYTOPLASMIC GRANULES
| Metachromatic granules (when stained with Loeffler’s Methylene Blue → Bluish purple
metachromatic color)
| a/k/a Polar bodies or Polymetaphosphates - reservoirs of energy & PO4 3- or Volutin
granules or Babes - Ernst granules
| Granules are abundant when the cells are present in nutritionally deficient conditions
| Stains used for granules -
1. Albert stain (MC)
2. Ponder’s stain
3. Neisser’s stain (not for Neisseria)
| Develop well on enriched media, Eg: Blood agar, Loeffler’s serum slope
| Also present in →
Personal Notes
` Gardnerella vaginalis (Bacterial vaginosis)
` Corynebacterium xerosis
| Granules --> stained more strongly gram positive than the rest of the bacilli
| Albert stain composition:
` Albert’s A solution - Toluidine blue, Malachite green, Glacial acetic acid, Ethyl alcohol
` Albert’s B solution (mordant or binding) - Iodine, Potassium iodide in water
CULTURE MEDIA
1. Enriched media - Loeffler’s serum slope (LSS)
` Advantage-
– Growth appears in 6 - 8 hrs
– Good for induction of granules
` Nutrient broth with horse or sheep serum + glucose - small colonies, circular, glistening,
whitish with a yellowish tinge
` Temperature : 15 - 40 degree Celsius
` Optimum pH : 7.2
2. Selective & Differential medium (Best) -
a. Potassium tellurite blood agar
1. Once Corynebacterium diphtheriae grows, it reduces Potassium tellurite (0.04 % )
present in the medium → Metallic potassium → Black colored colonies (also
produced by - C. ulcerans & C. pseudotuberculosis)
2. Disadvantage - Slow growth (24 - 48 hrs) or 2 days
Based on colony appearance on Potassium tellurite blood agar, 3 Biotypes of Corynebacterium
diphtheriae:
Gravis Intermedius Mitis
| Severity | grave or most | mildest disease
severe disease
| Colony | Daisy head | Frog’s egg | Poached egg
| Hemolysis On | Variable | Non-hemolytic | Usually hemolytic
blood agar
| Complications Haemorrhagic
| | Haemorrhagic | Obstructive lesion
Paralytic in the air passage
4. Other selective media -
a. Mcleod’s medium
b. Hoyle’s medium
BIOCHEMICAL REACTIONS
1. Hiss’s serum sugar media: Corynebacterium diphtheriae:
` Ferments both glucose and maltose (all biotypes)
` Starch is fermented → Gravis only
2 Microbiology
2. Urease test:
Personal Notes
` C. diphtheriae is Urease negative
` Urease positive → C. ulcerans & C. pseudotuberculosis
VIRULENCE FACTORS
Diphtheria toxin (DT) or Phage encoded toxin - Most important
| Exotoxin (protein ← mRNA ← gene)
| Gene is present on a phage → Corynephage or β - phage → when it attacks
C. diphtheriae → it transfers the gene for Diphtheria toxin (LYSOGENIC CONVERSION)
| Toxin mediated disease
| Optimum Iron concentration of 0.1 mg/L is needed for expression of DT gene
| Concentration of 0.5 mg/L inhibits expression of DT gene → upregulates DT repressor
gene present in bacterial chromosome
MOA of DT
| 2 subunits -
1. A (Active)
2. B (Binding) → host cell receptor (eg: Epidermal growth factor)
– Inhibit protein synthesis (translation) by ADP ribosylation of EF-2
– Toxin is inactive when it is released by the bacteria, as Active site on the A
subunit is masked
– Activation occurs by Proteases present in the infected tissues
– Toxin is produced by the bacteria at the site of infection (In Respiratory diphtheria
- most common is Faucial diphtheria) → Bacteria : localised, Toxin: acts both
locally and systemically (enters blood)
– Diphtheria → Toxaemia & not Bacteremia (\ Blood culture is Negative)
– Same MOA - also seen with Exotoxin - A of Pseudomonas aeruginosa
– Toxin can be inactivated or toxoided (treated with ether or formaldehyde) →
Toxoid (used as a vaccine) → Park william 8 strain
– DT concentration is expressed as Loeffler’s flocculating (Lf unit)
– 1 Lf unit - amount of toxin which flocculates most rapidly with 1 unit of anti
toxin
– Toxin has affinity for-
I. Myocardium
II. Adrenals
III. Nerve endings
Corynebacterium diphtheriae Part-1 3
Personal Notes
Gram positive Corynebacterium diphtheria bacteria,
which had been stained using the methylene blue
technique
4 Microbiology
Corynebacterium diphtheriae Part – 2
PATHOGENESIS & CLINICAL FEATURES OF DIPHTHERIA
Personal Notes
| Mode of transmission - Respiratory route
| Most common type - Respiratory diphtheria -most common site: Faucial region
| Most common type of diphtheria → Faucial diphtheria
| Incubation period: 3 - 4 days (2 - 6 days), may be as short as 1 day
| May be prolonged in carriers
| Definition: Localised infection of the mucous membrane and skin
| Rare types -
1. Laryngeal
2. Otitic
3. Nasal
4. Dermal or Cutaneous
5. Conjunctival
6. Genital (vulval or vaginal)
FAUCIAL DIPHTHERIA
| Bacteria are present in the throat → Diphtheria toxin (DT) is released → damage to the
epithelial cells → Inflammatory infiltrate in the throat → Dead bacteria, inflammatory
cells & epithelial cells, fibrin accumulation → Membrane in throat
| Membrane in throat
` Inner band of fibrin
` Surrounded by neutrophils, bacteria and RBCs
` a/k/a Pseudomembrane, adhered to mucosal base → ∴ Bleeds on removal → blocks
respiratory passage → Asphyxia
` Grayish white color
` 'Diphtheros' - greek word (leather - like)
Symptoms:
| Fever
| Sore throat
| Weakness
| Bull's neck appearance - massive tonsillar swelling and edema of the neck (Malignant or
Personal Notes
hypertoxic appearance)
| Foul breath
| Stridor
| Thick speech
Complications:
1. Detachment of the pseudomembrane → Blockage of the respiratory tract → Asphyxia
→ Death
2. Cardiogenic shock → Death
3. Myocarditis
4. Cardiomyopathy
5. Polyneuropathy
CUTANEOUS DIPHTHERIA
| Punched out ulcerative lesions with necrosis or rarely pseudomembrane formation
| Extremities
| Not d/t Toxin, it is d/t bacteria itself
| Caused by non-toxigenic strains
LAB DIAGNOSIS OF FAUCIAL DIPHTHERIA
I. Sample: Throat swabs (2 - 3) - gram stain, albert stain, culture
Note:
a. Throat may have commensals called as Diphtheroids (short, thick gram-positive bacilli)
b. Those strains of [Link] which may not have been lysogenically converted →
Pseudo carrier state (Harbouring non-pathogenic variant of an organism)
II. Gram stain: Gram +ve rods (3 - 6 µm), club shaped with cuneiform arrangement
III. Albert stain: Bacilli appear green, granules appear blue
IV. Culture
V. Toxigenicity tests:
1. In vivo (Animal inoculation) - guinea pigs
2. In vitro -
a. Elek's gel precipitation: [MCQ]
` Type of Immunodiffusion in gel described by Elek (1949)
Steps:
2 Microbiology
Precipitation
Personal Notes
Band
Antitoxin-soaked filter paper
Toxigenic Non-toxigenic
C. diphtheriae C. diphtheriae
` Once the agar solidifies, the test strain is streaked at 90 degrees to the filter paper
strip
` Incubate at 37 degrees celsius for 24 - 48 hrs
` Arrow head shaped precipitation band → test strain is toxigenic
` Also useful to know the relatedness between the strains isolated during an outbreak
of diphtheria --> the precipitate lines will fuse with each other if the toxins produced
by the stains are identical
b. Detection of TOX gene by PCR: Encodes for DT
c. Detection of Diphtheria toxin by ELISA or Immunochromatographic test
d. Tissue or cell culture cytotoxicity assay:
| If cell death occurs on treating live cells with culture filtrate → confirms toxigenic
strain
TREATMENT
| Mainstay of treatment → Antitoxin
| Toxoid is for long-term protection
| Antibiotics
1. Penicillin or Erythromycin → Drug of choice
2. Rifampicin or Clindamycin → Alternate drugs
| In Asphyxia → Emergency tracheostomy
| Vaccines
DIPHTHEROIDS
| These are species of genus Corynebacterium other than [Link]
| Commensals on skin & throat
| A/k/a Coryneforms
Corynebacterium diphtheriae Part – 2 3
Corynebacterium Diphtheroids Personal Notes
diphtheriae
Arrangement Cuneiform or Chinese Palisade arrangement (parallel stack)
letter appearance
Longer, slender Shorter, stouter
Staining Stains irregularly Stain more regularly or uniformly
Granules Has more number of Has less number of granules
granules
Medically Important Diphtheroids:
1. C. minutissimum -
` Localised skin infection k/a Erythrasma (axilla, groin)
` On Wood's lamp examination → Coral red fluorescence
2. [Link] -
` Infection (Sepsis) in immunocompromised (HIV infected) people
` Endocarditis
` Meningitis
` Lipophilic species
` MDR (multi drug resistant)
` Vancomycin is effective
3. C. parvum - Immunomodulator
4. [Link]-
` a/k/a PREISZ - NOCARD BACILLUS
` Pseudotuberculosis in sheep
` Suppurative lymphadenitis in horses
` Rarely infects humans
4 Microbiology