Lecture 9 Microbiology (Corynebacterium & Mycobacteria) 2021 PDF

Lecture 9 Microbiology: Corynebacterium & Mycobacteria 1  Content Microbiology: Mycobacteria & Corynebacterium 1. Mycobacterium tuberculosis The main biological characteristics of M. tuberculosis Pathogenesis of M. tuberculos...

Lecture 9 Microbiology: Corynebacterium & Mycobacteria 1  Content Microbiology: Mycobacteria & Corynebacterium 1. Mycobacterium tuberculosis The main biological characteristics of M. tuberculosis Pathogenesis of M. tuberculosis, Koch’s phenomenon Diagnosis of M. tuberculosis infection, treatment and prevention 2. Mycobacterium leprae & leprosy 3. C. diphtheria and infections 2  Corynebacterium diphtheriae Gram positive Strict aerobe Rods 0.5–1 µm in diameter and several micrometers long they possess irregular swellings at one end that give them the “club-shaped” appearance Granules irregularly distributed within the rod (often near the poles) cause deep staining with aniline dyes (metachromatic granules) that give the rod a beaded appearance Pleomorphic Pleomorphism = the occurrence of two or more forms in one life cycle 3  Specialized media Tellurite: Loeffler Black colonies Best colonial Not diagnostically morphology significant Dextrose horse serum tellurite inhibits many (1887) organisms but not C. now Dextrose beef diphtheriae serum 4  Culture On blood agar (pic. left), the C. diphtheriae colonies are small, granular, and gray with irregular edges and may have small zones of hemolysis. On agar containing potassium tellurite (pic.right), the colonies are brown to black with a brown-black halo because the tellurite is reduced intracellularly (staphylococci and streptococci can also produce black colonies). 5  Corynebacterium: Habitat: Skin of infected persons or normal carriers Upper respiratory tract GI tract Urogenital tract of humans wounds ❑ It is spread by droplets or by contact to susceptible individuals; ❑ the bacilli then grow on mucous membranes or in skin abrasions, and those that are toxigenic start producing toxin 6  Diphtheria member of normal flora of pharynx overgrowth upper respiratory tract pseudomembrane chocking bacteria do not spread systemically The toxin does disseminate 7  Corynebacterium: Pathogens C. diphtheriae - Diphtheria C.pseudotuberculosis - humans sheep, cattle, suppurative lymphadenitis C. ulcerans - humans pharyngitis cattle -mastitis C. haemolyticum – pharyngitis, cutaneous infection C. pyogenes - cattle, sheep, swine suppurative infection C.pseudodiphtheriticum - endocarditis 8  Diphtheria - Is a disease caused by the local and systemic effects of diphtherial toxin, a potent inhibitor of protein synthesis. The local disease is a severe pharyngitis typically accompanied by a plaque-like pseudomembrane in the throat and trachea. The life threatening aspects of dyphtheria are due to adsorption of the toxin across the pharyngeal mucosa and its circulation in the bloodstream. Multiple organs are affected, but the most important is heart, where the toxin produces acute myocarditis. 9  Pathogenesis Diphtheria toxin is absorbed into the mucous membranes and causes destruction of epithelium and a superficial inflammatory response. The necrotic epithelium becomes embedded in exuding fibrin and red and white cells so that a grayish “pseudomembrane” is formed—commonly over the tonsils, pharynx, or larynx. Any attempt to remove the pseudomembrane exposes and tears the capillaries and thus results in bleeding. The regional lymph nodes in the neck enlarge, and there may be marked edema of the entire neck, with distortion of the airway, often referred to as “bull neck” clinically. The diphtheria bacilli within the membrane continue to produce toxin actively. 10  Manifestation This child has diphtheria resulting in a thick gray coating over back of throat. This coating can eventually expand down through airway and, if not treated, the child could die from suffocation CDC 11  Diphtheria throat palate uvula tonsils 12  Clinical Findings When diphtheritic inflammation begins in the respiratory tract, sore throat and low-grade fever usually develop. Prostration and dyspnea soon follow because of the obstruction caused by the membrane. This obstruction may even cause suffocation if not promptly relieved by intubation or tracheostomy. Irregularities of cardiac rhythm indicate damage to the heart. Later, there may be difficulties with vision, speech, swallowing, or movement of the arms or legs. All of these manifestations tend to subside spontaneously. 13  Diphtheria Symptoms Pharyngitis Hypoxia Choking “Garritillo” = croup Fever (103 F) = 39,5 C Lymphadenitis All SIGNS & SYMPTOMS CAUSED BY TOXIN!!! 14  Diphtheria Pseudomembrane No True membrane Very few live cells Deposit of dead cells and protein Pseudomembrane can block the airway 15  Pharyngeal diptheria Inflammation similar to streptococcus throat Leucocytes infiltrated killed embedded in fibrin clot TOXIN!!!! 16  Corynobacteria COVERS Pseudomembrane tonsils CONTAINS uvula bacteria palate lymphocytes nasopharynx plasma cells larynx fibrin dead cells 17 Diphtheria Systemic complications Nerves toxic peripheral neuropathy paralysis of short nerves mouth, eye, facial extremities Cardiac Congestive heart failure High amount of toxin in hours Low amount of toxin 2-6 weeks 18  Diptheria toxin (DT) Exogenic toxin spreads systemic and fatal injury DT gene contained in the lysogenic phage DT causes myocarditis DT myocarditis may lead to congestive heart failure 19  Diphtheria toxin Diphtheria toxin is a heat-labile, single-chain, three domain polypeptide (62 kDa) that can be lethal in a dose of 0.1 µg/kg body weight. If disulfide bonds are broken, the molecule can be split into two fragments. Fragment B (38 kDa), which has no independent activity, is functionally divided into a receptor domain and a translocation domain. B unit binds to host cell A unit inhibits protein synthesis of eukaryotic cells Fragment A inhibits polypeptide chain elongation—provided nicotinamide adenine dinucleotide (NAD) is present—by inactivating the elongation factor EF-2 complex (ADP-ribosylation) 20  Virulence factors Diphtheria toxin !!! blocks protein synthesis Dermonecrotic toxin sphingomyelinase increases vascular permeability Hemolysin Cord factor Toxic trehalose 21  Virulence The “virulence” of diphtheria bacilli is attributable to their capacity for establishing infection, growing rapidly, and then quickly elaborating toxin that is effectively absorbed. C. diphtheriae does not need to be toxigenic to establish localized infection—in the nasopharynx or skin. Nontoxigenic strains do not yield the localized or systemic toxic effects. C. diphtheriae does not typically invade deep tissues and practically never enters the bloodstream. Over the last two decades invasive infections such as endocarditis and septicemia due to nontoxigenic C. diphtheriae have increased. 22  Wound or skin diphtheria Wound or skin diphtheria occurs chiefly in the tropics, although cases have also been described in temperate climates among alcoholic, homeless individuals, and other impoverished groups. A membrane may form on an infected wound that fails to heal. Absorption of toxin is usually slight and the systemic effects negligible. The small amount of toxin that is absorbed during skin infection promotes development of antitoxin antibodies. 23 Cutaneous diphtheria – produces alcerative lesions 24  Immunity Because diphtheria is principally the result of the action of the toxin formed by the organism rather than invasion by the organism, resistance to the disease depends largely on the availability of specific neutralizing antitoxin in the bloodstream and tissues. It is generally true that diphtheria occurs only in persons who possess no antitoxin antibodies (IgG) (or less than 0.1 IU/mL) Antibodies neutralize toxin Formalin treatment of toxin produces toxoid, which retains its antigenicity but not toxicity of naïve toxin and is used in immunization the disease 25  Testing immunity Schick skin test – toxin 26  Prevention Prevention may be achieved through immunization 3-4 doses of diphtherai toxoid produce immunity by stimulating anti-toxin production. The initial series is begun in the 1st year of life. Booster immunization at 10 years intervals maintain immunity. Fully immunized individuals may become infected with C.dyphtheria because the antibodies are directed only against toxin, but the disease is mild. 27  Treatment Anti-toxin – aims neutralizing of free toxin Antibiotic (antibacterial therapy) - erythromycin is the most effective 28  Immunization against diphtheria (infant) Disease vanished in US – without immunization will return toxoid (+ pertusis and tetanus) DPT neutralizing antibodies colonization not inhibited – found in normal flora 29  Diphtheria Laboratory diagnosis Speedy diagnosis Differentiate from commensals “diphteroids” nose & throat C. xerosis C. hofmanni Throat swabs (confirmatory) Blood Tellurite Virulence test 30  Diagnostic C. diphtheriae should not be confused with: diphtheroids – other corynebacteria – propionibacteria 31 Robert Koch Koch’s postulates: Organism consistently isolated from diseased individuals Organism cultivated in pure form Signs and symptoms induced after inoculation Same organism isolated from experimentally infected individual 32  Mycobacterium tuberculosis obligate aerobe non-motile acid-fast rods do not form spores 33  Mycobacterial diseases tuberculosis-like leprosy-like 34  Laboratory diagnosis M. tuberculosis acid fast bacteria – sputum Acid fast: Bright red to intensive purple (B), Red, straight or slightly curved rods, occurring singly or in small groups, may appear beaded Non-acid fast: Blue color (A) 35  Differential Staining Methods - Acid-Fast Staining Acid-fast Stain: ACID-FAST Cell Color Cell color Mycobacterium and many STAIN Nocardia species are called acid-fast because during an Procedure Reagent Acid fast Non acidfast acid-fast staining procedure bacteria bacteria they retain the primary dye Primary dye Carbolfuchsin RED RED carbol fuchsin despite Decolorizer Acid-alcohol RED COLORLESS decolorization with the powerful solvent acid-alcohol. Couterstain Methylene blue RED BLUE Nearly all other genera of bacteria are nonacid-fast. The acid-fast genera have lipoidal mycolic acid in their cell walls. It is assumed that mycolic acid prevents acid-alcohol from decolorizing protoplasm. The acid-fast stain is a differential stain. 36  Cell wall structure The cell wall structure of Mycobacterium tuberculosis deserves special attention because it is unique among prokaryotes, and it is a major determinant of virulence for the bacterium. The cell wall complex contains peptidoglycan, but otherwise it is composed of complex lipids (>60% of the total cell mass). Long chains of fatty acids called mycolic acids Lipoarabinomannan (LAM) – a lipid polysaccharade complex extending from the plasma membrane to the surface. The waxy coat makes the cell impermeable and hydrophobic, which is determining acid fastness (once stained difficult to decolorate). 37 38 Laboratory diagnosis M. tuberculosis (culture) grows very slowly – two weeks or longer – non-pigmented colonies – niacin production differentiates from other mycobacteria polymerase chain amplification – rapid diagnostic 39  Tuberculosis (TB, consumption) M. tuberculosis major human disease – healthy people (one third of the world population is infected) problems – association with AIDS – multiple drug-resistance 40  Tuberculosis: epidemiology Infection of the 18th and 19th century Attack rates still high in many developing countries The disease has major sociologic components, flourishing with ignorance, poverty, poor hygiene Globally 1/3 of world population is infected 41  Celebrities who died from tuberculosis Kamala Nehru 37 y. Vivien Leigh 54 y – Franz Kafka 41 y - the Anton Chekhov 44y - the wife of the British actress, German novelist. His – The Russian writer Jawaharlal Nehru - who performed a role famous work is "The the first Prime of Scarlett O'Hara in Metamorphosis" Minister of India and Gone with the Wind the mother of Indira (1939) 42 Gandhi  Interesting reading At the beginning of the XX century, half of Western Europe had faulty lungs. CristinaVilaplana (2017). “A literary approach to tuberculosis: lessons learned from Anton Chekhov, Franz Kafka, and Katherine Mansfield”, International Journal of Infectious Diseases Volume 56, March 2017, Pages 283-285. https://doi.org/10.1016/j.ijid.2016.12.012 Summary: Letters by notable writers from the past century can provide valuable information on the times in which they lived. In this article, attention is drawn to the lessons learned from three famous writers who died of tuberculosis: Anton Chekhov, Franz Kafka, and Katherine Mansfield. The characteristics of the course of the disease in the pre-antibiotic era and the importance of addressing mental health in the management of tuberculosis are discussed. 43  Transmission Most infections are by respiratory route Repeated coughing generates infectious dose (ID50