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)
Tuberculous or Non-Tuberculous Mycobacteria:
Tuberculous Mycobacteria
M. leprae
M. tuberculosis complex (MTBC)
Non-Tuberculous Mycobacteria (NTM)
Other names = Atypical Mycobacteria
Environmental Mycobacteria (EM)
What is Tuberculosis?
A disease caused by Mycobacterium tuberculosis complex in humans
Spread primarily through aerosols to infect the lungs
Pulmonary tuberculosis manifests as a chronic cough
producing blood and sputum, impaired breathing, fever, night sweats and weight loss
Extrapulmonary tuberculosis can also occur in the nervous system causing meningitis, the circulatory and lymphatic
systems, GU and GTI tracts, and in bones and joints
Mycobacterium tuberculosis Complex
The Mycobacterium tuberculosis complex (MTBC) contains the species
M. tuberculosis
M. africanum
M. bovis
Prevention of M. tuberculosis
Live Bacille Calmette-Guérin (BCG) vaccine
In children:
- Small protective effect against M. tuberculosis infection
(risk ratio (RR) of 0.81)
- Significantly higher protection against progression
from infection to TB disease (RR of 0.42)
- Highly protective effect against meningeal TB (RR of 0.1)
and miliary TB (RR of 0.08)
Produces false positives in the TST (tuberculin skin test)
Diagnosis of M. tuberculosis
Clinical symptoms/signs of the disease
Chest X-ray
Tuberculin-skin test (TST)
Microscopic and culture examination of sputumNucleic Acid Amplification Technique (NAAT)
Interferon Gamma Release Assay (IGRA)
Discovery of Drugs for the Treatment of TB
1943: Streptomycin
1948: p-aminosalicylic acid
1952: Isoniazid (INH)
1954: Pyrazinamide (PZA)
1955: Cycloserine
1957: Kanamycin
1960: Ethionamide
1961: Ethambutol (EMB)
1963: Rifampicin (RMP)
Treatment of DS-TB
Recommended therapy for Active TB cases
Patients with Drug-Susceptible (DS) TB
Intensive phase 2 months INH, RMP, PZA, EMB+
Continuation phase 4 months INH, RMP
Isoniazid
Inhibits InhA which is essential for mycolic acid synthesisBactericidal activity (to growing M. tuberculosis)
Main adverse effects: hepatotoxicity; peripheral neuropathyInteractions with: anti-epileptics
Rifampicin
Inhibits RpoB of RNA Polymerase which is essential fortranscription
Bactericidal activity
Main adverse effects: hepatotoxicity; GI disturbance; rashInteractions with: HAART; oral contraceptive pill; corticosteroids
