Week 7 Flashcards
GENERAL CHARACTERISTICS Corynebacterium diphtheriae
- Gram-positive, pleomorphic, straight or slightly curved, thin rods with tapered or club-shaped ends
- Arranged: singly; in palisades of parallel cells; in pairs not fully separated, laying at sharp angles to each other (V, L, Y formations); groups may take on appearance of Chinese characters* Not acid-fast, do not branch
- Non-motile, do not form spores
- Metachromatic / Babes-Ernst granules (accumulations of
polymerised polyphosphates) within cells common - Cell wall contains meso-diaminopimelic acid, most have short-chain mycolic acids (corynomycolic acids)
- Aerobic, facultatively anaerobic
- Catalase positive
- Ferment glucose and other carbohydrates
- Some species require lipids for optimal growth
Corynebacterium diphtheriae NATURAL HABITAT
- Widely distributed in nature, commonly found in soil and water
- Reside on skin and mucous membranes of humans and animals
- Corynebacterium spp. (except for Corynebacterium diphtheriae)
usually considered contaminants when recovered in clinical laboratory - Repeated isolation of Corynebacterium spp. from normally sterile sites suggests that organism may be cause of infectious process
- Corynebacterium diphtheriae most often isolated from nasopharynx or skin lesions of patients with diphtheria
- Corynebacterium diphtheriae can be carried on mucous membranes and skin of healthy humans (particularly non-toxigenic strain), not found in animals
Corynebacterium diphtheriae VIRULENCE FACTORS - TOXIN
- Major biochemical determinant in pathogenesis of infection which accounts for all of the systemic pathological effects
- Produced only by strains of Corynebacterium diphtheriae infected with a temperate bacteriophage carrying structural gene for toxin
production - Non-toxigenic strains may be converted to a lysogenic, toxigenic state by infection with suitable tox+
corynephage - Synthesised as a single polypeptide consisting of:
−fragment B (binding) at carboxyterminal end, attaches toxin to host cells (or to any eukaryotic cell)
−fragment T (translocation), inserts toxin into host cells
−fragment A (active) at amino-terminal end, toxic fragment - Only formed after uptake of toxin into cell (by protease cleavage and reduction of disulphide bonds)
- Inactivates elongation factor-2 (EF-2) by ADP ribosylation and
thereby, inhibits protein synthesis - Prokaryotic and mitochondrial protein synthesis not affected because of different elongation factor involvement
- Stable, single molecule can kill cell (bacterium produces 5,000
molecules per hour) - Myocardial and peripheral nerve cells are particularly susceptible to the activity of the toxin
- Animals vary in susceptibility: − humans and experimental rabbits, guinea pigs are very susceptible−rats, mice are much less susceptible
Corynebacterium diphtheriae TOXOID and ANTITOXIN
TOXOID
* Addition of low concentrations of formaldehyde (0.3% formalin) to diphtheria toxin and incubation at 37oC destroys its toxicity
* The toxoid formed cannot be cleaved into A and B fragments, lacks ADP ribosylating activity, and does not bind to cell membranes
ANTITOXIN
* Both the A and B moieties contain a number of antigenic determinants and generate an immune response in humans and animals
* The anti-toxin generated acts by competing with toxin for surface receptors on sensitive eukaryotic cells
* Prevents binding of free toxin circulating in blood to undamaged cells
VIRULENCE FACTORS - INVASIVENESS C. diphtheria
- K antigen
− heat-labile proteins
−localised in superficial layers of cell wall
−responsible for type specificity of Corynebacterium diphtheriae - Cord factor
−toxic glycolipid
− pharmacological activity similar to cord factor from
Mycobacterium tuberculosis
−causes disruption of mitochondria and reduction of respiration - Neuraminidase and N-acetylneuraminate lyase
−enzymes
− degrade the sialic acid, N-acetylneuraminic acid, located on the surface of host cells
− provide readily available source of energy for bacteria colonising mucous membranes
CLINICAL SIGNIFICANCE - CORYNEBACTERIUM DIPHTHERIAE
- Causative agent of diphtheria
- Acute communicable disease manifested by: − local infection of upper respiratory tract − systemic effects of toxin, most notable in heart and peripheral nerves* Humans are the only natural hosts of Corynebacterium diphtheriae* Symptom-free carriers and persons in incubation stage of disease are major sources of most infections
- Organisms transmitted from person to person (directly or indirectly)* Transmission via droplet infection is a major mechanism of transfer in respiratory disease
- Discharges from extrarespiratory sites e.g. skin ulcers, can provide a source of pharyngeal and cutaneous disease
- Incubation period of 1 to 7 days
- Initial lesion occurs on tonsils and oropharynx, may spread to
nasopharynx, larynx, trachea - Organisms multiply rapidly on epithelial cells, producing exotoxin that causes necrosis of cells in area and an inflammatory reaction
accompanied by outpouring of fibrinous exudate - A tough adherent pseudomembrane forms
Respiratory disease
* clinical manifestations vary depending on:
− virulence of organism
− host resistance
− anatomical location of lesion
* in tonsilar diphtheria (most common clinical presentation) - abrupt onset characterised by low-grade fever, malaise, sore throat
* cervical lymph nodes oedematous and tender (“bull neck” appearance)
* extension to larynx and trachea results in severe form of disease
* mechanical obstruction of airway by pseudomembrane and accompanying oedema introduce risk of suffocation
* high mortality rate whereby death can result from congestive heart failure and cardiac arrhythmias caused by toxic myocarditis
Extrarespiratory disease
* Primary or secondary lesions may occur in other parts of body
* Most common site is the skin
* In rare cases, conjunctiva, cornea, ear, vagina can be infected
* Lesions appear at site of minor abrasions and appear as chronic, spreading, non-healing ulcers covered with greyish pseudomembrane
* Streptococcus pyogenes or Staphylococcus aureus may also be present in lesion
COLLECTION, TRANSPORT, AND STORAGE OF SPECIMENS C. diphtheriae
- if diphtheria suspected - swab from inflamed areas of
pseudomembranes formed in throat and nasopharynx - suspected carriers - swab
- material from wounds - removed by swab or aspiration
- specimen immediately transported to laboratory or inoculated onto proper media
- if not possible, should be sent to reference laboratory by being shipped dry in sterile tube
CULTURE MEDIA C. diphtheriae
Selective media
* Modified Tinsdale medium
* Cystine-tellurite (CT) blood agar medium
* Both media contain potassium tellurite that causes C. diphtheriae to appear black on agar due to tellurite reductase activity
Non-selective media
* Loeffler serum medium
* 5% sheep blood agar medium
INCUBATION CONDITIONS C. diphtheriae
- 35oC to 37oC
- for 24 to 48 hours
- with or without added CO2
DIRECT EXAMINATION C. diphtheriae
- Gram-stained smears
−for observation of coryneform morphology - Neisser- or Albert-stained smears −for observation of metachromatic granules
- PCR assay −for direct detection of toxin gene in clinical specimens and pure cultures
IDENTIFICATION C. diphtheriae
- MALDI-TOF MS
- API Coryne strip (bioMérieux Vitek)
- Colony morphology
- Gram stain of colony
- Neisser or Albert stain of colony
- Urease production
- Nitrate reduction
- Immunochromatographic Strip (ICS) Test
ANTIBIOTIC SUSCEPTIBILITIES -
CORYNEBACTERIUM DIPHTHERIAE
- Penicillin or erythromycin recommended to:
−eliminate organism from respiratory tract in disease or carrier state
−stop toxin production
−relieve local infection
− prevent spread - administration of diphtheria antitoxin (intramuscularly or
intravenously in single dose) in adequate amounts only specific and effective treatment - antitoxin should be administered as soon as presumptive
diagnosis of diphtheria made clinically - antitoxin neutralises only toxin not already bound to target cells
IMMUNISATION C. diphtheriae
Active immunisation
* key to control and prevention
* toxoid administrated as aluminium salt-adsorbed preparation
* primary course of immunisation should be started during first 8 weeks of life
* consists of 0.5mL doses of toxoid combined with tetanus toxoid and pertussis vaccine (DTaP) given intramuscularly
Passive immunisation
* immunisation with 5,000 to 10,000 units of antitoxin employed for protection of non-immunised persons who were exposed to toxigenic organisms
Phenotypic Properties of Mycobacteria
Rod-shaped
Acid-fast cell wall
Non-spore forming
Aerobic
Aflagellate
Fast versus Slow Growers Mycobacteria
Mycobacteria can be divided into 2 main groups onthe basis of their growth rates in vitro:
“FAST” = form readily visible colonies on solid growthmedia within 7 days
“SLOW” = require longer than 7 days to producereadily visible colonies on solid growth media
Fast-Growing Mycobacteria that are capable of causingNTM infections in humans consist primarily of thefollowing 3 groups:
Mycobacterium fortuitum
Mycobacterium chelonae-abscessus
Mycobacterium smegmatis
Slow-Growing Mycobacteria that cause disease inhumans include the following:
Mycobacterium avium complex (MAC)
Mycobacterium leprae
Mycobacterium tuberculosis complex (MTBC)
