Lec3_Bact_Non_Spore_Forming_Gram_Positive_Rods_Corynebacterium_Nov PDF

Non-Spore-Forming Gram-Positive Rods Corynebacterium diphtheriae Dr. Nada A. Abdelrahim  3 important pathogens in this group: ❖Corynebacterium diphtheriae ❖Listeria monocytogenes ❖Gardnerella vaginalis Corynebacterium diphtheriae Disease C. diphtheriae causes Diphtheria Oth...

Non-Spore-Forming Gram-Positive Rods Corynebacterium diphtheriae Dr. Nada A. Abdelrahim  3 important pathogens in this group: ❖Corynebacterium diphtheriae ❖Listeria monocytogenes ❖Gardnerella vaginalis Corynebacterium diphtheriae Disease C. diphtheriae causes Diphtheria Other Corynebacterium species (Diphtheroids) are implicated in Opportunistic Infections Coryneform bacteria demonstrate similar morphology to that of Corynebacterium spp.  Important Properties Corynebacteria are Gram-positive Rods, Club shaped (i.e. wider at one end), arranged in Palisades or in V- or L-shaped formations (see picture ) Characteristic Chinese Letter Arrangement Rods have beaded appearance (consist of granules of highly polymerized polyphosphate—a storage mechanism for high-energy phosphate bonds) Granules stain metachromatically (i.e., a dye that stains the rest of the cell blue will stain the granules red - Albert’s Stain - when grown on Loffler’s Serum) In Albert’s Stain: granules appear purple-black against the light green cytoplasm Non-motile, Non-capsulate  Transmission Humans are the Only Natural Host Both toxigenic and nontoxigenic organisms reside in the upper respiratory tract (organism is maintained in the oroparynx or skin of asymptomatic carriers) Transmitted by airborne droplets (spread directly from person to person) It can also infect the skin at the site of a preexisting skin lesion (occurs primarily in the tropics, but can occur worldwide in persons with poor skin hygiene) To limit contact with diphtheria bacilli to a minimum, patients with diphtheria should be isolated  Pathogenesis Exotoxin production is essential for Pathogenesis, Invasiveness is Also Necessary (because the organism must first establish and maintain itself in the throat) Diphtheria toxin inhibits protein synthesis by ADP-ribosylation of elongation factor-2 (EF-2) Toxin affects all Eukaryotic cells (regardless of tissue type) but has no effect on analogous factor in prokaryotic cells  Diphtheria Exotoxin: is single polypeptide with two functional domains The binding (B) domain: mediates binding of the toxin to glycoprotein receptors on cell membrane The active (A) domain: possesses enzymatic activity that cleaves nicotinamide from nicotinamide adenine dinucleotide (NAD), and transfers the remaining ADP-ribose to EF-2, thereby inactivating it DNA codes for diphtheria toxin is part of the DNA of a temperate bacteriophage (called Beta Phage) During lysogenic phase of viral growth, the DNA of this virus integrates into the bacterial chromosome and the toxin is synthesized C. diphtheriae cells that are not lysogenized by this phage Do Not produce exotoxin and are nonpathogenic  Host Response to toxigenic C. diphtheriae infection consists of the following: (1) A local inflammation in the throat, with a fibrinous exudate that forms the characteristic tough, adherent, gray pseudomembrane (2) Antibody that can neutralize exotoxin activity by blocking the interaction of the binding domain with the receptors, thereby preventing entry into the cell  Immune Status of a person can be assessed by Schick’s test: - Intradermal injection of 0.1 mL of purified standardized toxin - If patient has no antitoxin, the toxin will cause inflammation at the site 4 to 7 days later - If no inflammation occurs, antitoxin is present and the patient is immune The test is rarely performed in the United States except under special epidemiologic circumstances  Clinical Findings Clinical Diseases: 1\ Respiratory diphtheria: Incubation period is 2-6 days Inflammation begins in the Respiratory Tract, causing Sore Throat, Exudative Pharyngitis that develops into Pseudomembrane, and Low-Grade Fever Prostration and Dyspnea soon follow, which may lead to Suffocation if not promptly relieved by Intubation or Tracheotomy Damage to the heart causes irregular cardiac rhythm Visual disturbance, difficulty in swallowing and paralysis of the arms and legs also occur but usually resolve spontaneously Death may be due to asphyxia or heart failure Intubation Tracheotomy  The most prominent sign of diphtheria is the thick, gray, adherent pseudo- membrane over the tonsils and throat Other clinical findings in diphtheria are nonspecific: - Fever (low grade), Sore Throat, Cervical Adenopathy 3 prominent complications: (1) Extension of the membrane into the larynx and trachea, causing airway obstruction (2) Myocarditis accompanied by arrhythmias and circulatory collapse (3) Nerve weakness or paralysis, especially of the cranial nerves Paralysis of the muscles of the soft palate and pharynx can lead to regurgitation of fluids through the nose Peripheral neuritis/neuropathy affecting the muscles of the extremities also occurs  Bull-neck appearance Diphtheria Pseudomembrane  2\ Cutaneous diphtheria: causes ulcerating skin lesions covered by a gray membrane Lesions are often indolent and do not invade surrounding tissue Systemic symptoms rarely occur In countries like the US, cutaneous diphtheria occurs primarily in poor communities Laboratory Diagnosis Involves both: isolating the organism & demonstrating toxin production The decision to treat with antitoxin is clinical, cannot wait for lab results A throat swab should be cultured on: ❖ Loeffler’s Serum Slope Agar, Tellurite Blood Agar, and Blood Agar Plates The tellurite plate contains a tellurium salt that is reduced to elemental tellurium within the organism The typical gray-black color of tellurium in the colony is a diagnostic criterion  If C. diphtheriae is recovered, Animal Inoculation or Antibody-Based Gel Diffusion Precipitin Test should be performed to detect toxin production PCR assay for detection of toxin gene (tox) in clinical isolates can also be used Toxigenicity tests: 1. in vivo test: inject the culture into antitoxin-protected and unprotected guinea pigs subcutaneously 2. Tissue culture neutralization assay 3. in vitro test: immunodiffusion assay (Elek test ) 4. Detection of toxin gene by PCR  Diphtheria toxigenicity tests determine if a strain of Corynebacterium diphtheriae produces toxins The most common in vitro test for diphtheria toxigenicity is Elek Test (the gold standard for confirming toxin production) Elek Test: uses immunodiffusion to detect the development of an immunoprecipitin band on filter paper placed over an agar culture of the bacteria A modified version of the test can produce accurate results in 16 hours, compared to 48 hours for the conventional test Elek Test  Smears of throat swab should be stained with both Gram stain and methylene blue Diagnosis of diphtheria cannot be made by examination of smears But, smear findings of many tapered, pleomorphic Gram positive rods can be Suggestive Methylene blue stain is excellent for revealing the typical metachromatic granules Treatment Treatment of choice is: Antitoxin Given immediately on clinical impression (because of delay in lab results) The toxin binds rapidly and irreversibly to cells and, once bound, cannot be neutralized by antitoxin The function of antitoxin is, therefore, to neutralize unbound toxin in the blood Because antiserum is made in horses, the patient must be tested for hypersensitivity, and medications for the treatment of anaphylaxis must be available Serum sickness may occur after administration of antiserum made in horses  Treatment of diphtheria: - Prompt administration of Diphtheria Antitoxin - Antibiotics: Penicillin G (single dose/intramuscular) or Erythromycin (course/oral) For 14 days for individuals presenting with symptoms of diphtheria (NOT a substitute for antitoxin) - Maintenance of an open airway - Treatment of bacteremia or endocarditis must be guided by antibiotic susceptibility tests Role of Antibiotics: Inhibit growth of organism Reduce toxin production, and Decrease incidence of chronic carriers  Prevention Diphtheria is very rare (in countries like the US) because children are immunized with Diphtheria Toxoid Diphtheria Toxoid: usually given as combination of Diphtheria Toxoid, Tetanus Toxoid, and Acellular Pertussis Vaccine, abbreviated as DTaP Diphtheria toxoid: prepared by treating the exotoxin with formaldehyde (inactivates toxic effect but leaves antigenicity intact) 3 doses given at 2, 4, and 6 months of age, with boosters at 1 and 6 years of age Because immunity decreases, booster every 10 years is recommended Immunization Does Not prevent nasopharyngeal carriage Prophylactic antibiotic treatment to unimmunized contacts  Other Corynebacterium Species Ubiquitous in plants and animals Many are found as part of human normal flora and may cause opportunistic infections (such as pneumonia, endocarditis, and soft tissue and bone infections, in immunocompromised patients) C. jeikeium: sepsis, endocarditis, wound infections, foreign body infections C. urealyticum: causes UT infections (It is strong urease producer, infection of UT may lead to formation of stones) C. ulcerans: closely related to C. diphtheriae (may cause diphtheria-like disease) Resistant to many antibiotics. Treatment of bacteremia or endocarditis must be guided by antibiotic susceptibility tests  Upon entering the cell, the organism produces listeriolysin (allows it to escape from phagosome into the cytoplasm -thereby escaping destruction in phagosome) Because Listeria preferentially grows intracellularly, cell-mediated immunity is a more important host defense than humoral immunity Suppression of cell-mediated immunity predisposes to Listeria infections L. monocytogenes can move from cell to cell by means of Actin Rockets/ Comet Tails (filaments of actin polymers) and propel bacteria through membrane of one human cell and into another (see next slide) Listeria can also move using flagella at temperatures of 30°C and below. But at 37°C, Listeria moves primarily using actin rockets