Microbio Lec 8 - Mycobacteria

MICROBIOLOGY LECTURE 8 – Mycobacteria Notes from Lecture USTEMED ’07 Sec C - AsM Characteristics: aerobic, slightly curved or straight rods 0.2 to 0.6 um wide; 1.0 to 10 um long cell wall: multilayered complex lipids common properties with Corynebacterium and Nocardia: o produce mycolic acid o ~guanine + cytosine content (G+C) Mycobacterium tuberculosis Tuberculosis An ancient disease Seen in stone age skeletons and early Egyptian mummies 1882 – Koch’s discovery Koch’s postulates: o An organism associated with clinical disease o Isolated it in pure culture o Reproduces the disease in animals o Recovered the bacillus in pure culture from the experimental animal Morphology of MTb Slender, straight or slightly curved rod with rounded ends 0.3 to 0.6 um width x 1-4 um length true branching in old cultures and smears from caseous lymph nodes acid fast: lipid-barrier (mycolic acid) and carbolfushin dye complex hydrophobic surface layers trap the dye Mycobacterial Cell Wall 60% lipid including mycolic acids backbone: covalent structure consisting of 2 polymers covalently linked by phosphodiester bonds: o peptidoglycan o arabinogalactan MTb Physiology Cultural characteristics: o Strictly aerobic o Most spp. Grow slowly with generation times 8 to 24 hrs. o Grow fairly on simple artificial media except M. leprae Species are differentiated by: o Rate and optimal temperature of growth o Production of pigments o Biochemical tests (niacin, catalase test, urease test, etc) Antigenic Structure of Mycobacteria 1. Old tuberculin (OT) o 1881 – described by Koch by boiling a 6-week old broth culture o Heat–stable protein is the active component 2.

Purified Protein Derivative (PPD) o Partially purified preparation of OT prepared by ammonium sulfate fractionation o PPD-S: adopted by WHO for skin testing

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Purified Antigens: o Recombinant DNA techniques & antigen expression in E.coli o Affinity purification of antigen preparations using monoclonal antibody immunosorbent columns

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Polysaccharides: o Protein-free polysaccharides (arabinogalactans & arabinomannans) i. Immunogenic ii. Give precipitin reactions with antisera iii. Active in C’fixation & hemmaglutination reactions

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Phosphatidyl Inositol Mannosides (PIMS) o Lipoteichoic acid-like polymers with a role in macrophage recognition o Associated with cross protective immunity

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Other immunoreactive components a. Wax D & muramyldipeptide (MDP)  peptidoglycans (cell wall)  adjuvant activity  induce a cell-mediated immune response against the protein b. Trehalose-6-6’-dimycolate  cord factor  immunoreative properties  adjuvant activity  elicits extensive pulmonary granulomas  anti-tumor properties c. Sulfatides (Trehalose 2’-sulfates esterified with fatty acids)  sulfur-containing glycolipids (sulfolipids)  can replace cord factor as a component of an oil-BCG cell wall or endotoxin preparation causing tumor regression

Determinants of Pathogenicity Cord factor Sulfatides Epidemiology of MTb TB is a global problem 8 to 10 million new cases worldwide 3 million deaths worldwide Yearly decline in TB incidence ended in 1984 when the HIV infection increased in number Philippine statistics: FHSIS-DOH 2001 o Respiratory TB: 6th leading cause of morbidity: 11-,841 cases and rate of 142.2/100,000 population o TB meningitis: 466 cases and rate of 0.6/100,000 population o Other forms of TB: 11,494 cases and rate of 14.7/100,000 population Transmission o Inhalation of dried residues of droplets containing tubercle bacilli o Droplet nuclei (1 to 10 um) can reach the alveoli and initiated infection Tuberculous infection vs. Tuberculous disease: Tuberculin Clinical test symptoms Tuberculous infection + Tuberculous disease + + -

risk factors for the development of Tuberculous disease: o intrinsic characteristics of the individual – age, sex, body build, & genetic susceptibility o use of adenocorticosteroids & immnunosuppressive agents o hematologic diseases o reticuloendothelial disease o Diabetes mellitus o Silicosis o HIV infection

Pathogenesis of TB Inhaled MTb bacilli

alveoli

Multiply in the pulmonary epithelium or macrophages 2-4 weeks Bacilli are destroyed by immune system

Survivors blood Extrapulmonary sites Bacterial sulfolipids inhibit fusion of phagocytic vesicles with lysosojmes

Clinical Manifestations Primary Tuberculosis o No previous contact with the organisms o 95% of cases are arrested o positive tuberculin test o chest x-ray: pulmonary nodule & some fibrosis (Ghon complex) o 10% develop clinical Tb later in life Primary disease: 1. Initial phase o Primary Tb  – mild or asymptomatic and results in exudative lesions with PMN and fluid accumulation around the bacilli  cell-mediated immunity and hypersensitivity rxn 2. tubercle formation o granulomatous lesion

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central area of large, multinucleate giant cells (macrophage syncytia) with tubercle bacilli, an id-zone of pale epithelioid cells, and a peripheral collar of fibroblasts and mononuclear cells

o o o o o -

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Alcoholism Advanced age Severe stress Immunosuppressive tx DM, AIDS

Chronic Pulmonary Tuberculosis o Insidious onset of fever, fatigue, anorexia, night sweats, and wasting o Cough and sputum o Hemptysis and chest pain Extrapulmonary Tuberculosis o Miliary lesions in the bones, joints, GIT, meninges, lymph nodes and peritoneum o AIDS patients with Tb progress into severe and unusual manifestations

Tuberculin Test delayed hypersensitivey to M. tuberculosis protein antigens Mantoux test: PPD o 0.1 mL of 5 TU of PPD-S o Activity is expressed as tuberculin units (TU)

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Intradermal injection (48-72 hrs) induration o Positive reaction 4-6 weeks after initial contact with bacilli Tine Test: multiple puncture method o For screening only

Primary Tuberculosis

Lesion breaks down

Lesion arrest

Fibrosis & cacification

Viable, nonproliferating organisms

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Liver, spleen, kidneys, bone, meninges

Miliary Tb

Caseous matrials Cavity formation Spread of infection -

Interpretations of the Mantoux skin test for Tuberculosis

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Mantoux test (tuberculin skin test)

Bacilli is dispersed in the lymph and blood stream

Stages in the Pathogenesis of tuberculosis

Reactivation of Tuberculosis Due to: o Impairment in immune status o Malnutrition

Colonies of M. Tb on Middlebrook 7H11 agar viewed microscopically. Note the beginning of the cording characteristics

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Conditions affecting the tuberculin reaction: o Negative – active TB in suppressed cellmediated immunity o Cross-reaction may be observed with other spp. of Mycobacteria

Colonies of M. tuberculosis (3 to 4 weeks old) on Middlebrook 7H11 agar. Colonies have a rough appearance and exhibit cording, exemplified by the darker areas

Laboratory Identification Identification of M. tuberculosis in clinical specimens: o Ziehl-Neelsen stain o Kinyoun stain

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Fluorescent acid fast dye (auraminerhodamine) of sputum, bronchial washings, urine, spinal fluid sediment, biopsy material

M. Tuberculosis colonies on Lowenstein-Jenssen agar 8 weeks of incubation

M. tuberculosis stained with Kinyoun acid-fast stain

M. tuberculosis stained with fluorochrome stain

1. 2.

Molecular techniques: Amplified MTb direct test o makes copies of 16S ribosomal RNA and detected by genetic probe PCR o amplifies small portion of target DNA o facilitates DNA finger printing of specific strains

Laboratory Report of Acid Fast Bacilli Number of Bacilli Report 0 No AFB seen 1-2/300 fields Doubtful; request another specimen 1-9/100 fields + 1-9/10 fields ++ 1-9/field +++ >9/field ++++ Culture of MTb Lowenstein –Jensen (L-J) medium o egg- potato- base media Middlebrook 7H-10 o agar- base media 5% to 10% CO2 3-6 weeks incubation

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12 B vial for Bactec MTb 460- radiometric and semiautomated method of TB culture

M. Tuberculosis on L-J agar slant

M. Tb on Middlebrook 7H11 agar (with casein hydrolysates): cream colored, dry and wrinkled

Treatment of MTb Multidrug therapy: First line drugs o Isoniazid o Rifampin o Ethambutol o Streptomycin o Pyrazinamide Multidrug resistance (MDR) Drugs used in the treatment of tuberculosis Drug Daily Adult Dosage Major Toxicity First-line agents Isoniazid Rifampin

300 mg orally or im 600 mg orally or iv

Hepatitis, neurophathy Hepatitis, flulike syndrome

Pyrazinamide

1.5-2.5 g orally

Hepatitis, hyperuricemia

Ethambutol Streptomycin

15-25 ng/kg orally 0.5-1 g im

Optic neuritis Vestibular dysfunction, deafness, renal (rare)

Cycloserine

250-500 mg twice a day orally

Seizures, psychiatric symptoms, CNS dysfunction

Ethionamide

250-500 mg twice a day orally

Nausea, vomiting, hepatitis psychiatric symptoms

Capreomycin

0.5-1g im, then 1 g 2-3 times a day

Deafness, vestibular dysfunction, renal damage

Second line agents

Kanamycin Prevention of MTb INH prophylaxis o No protection to uninfected person after treatment is stopped BCG vaccination o Useless after the patient has been infected with tubercle bacilli Mycobacterium bovis

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causes clinical illness similar to that caused by M. tuberculosis milk is the common vehicle primary lesion in the cervical or intestinal lymph nodes

Mycobacteria other than Tuberculosis (MOTT) -

non-tuberculous Mycobacteria (NTM) Runyon Classification of NTM:

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Photochromogens pigment following light

– develop exposure to

Scotochromogens M. scofulaceum o chronic cervical adenitis in children o resistant to antituberculous drugs o surgical excision of infected cervical nodes o habitat are raw milk, soil, water, dairy products M. szulgai o Cervical adenitis in children o Habitat is water and soil Scotochromogen M. gordonae with yellow colonies

Photochromogens – unpigmented when grown in the dark (A) and develop pigment after light exposure (B)

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Scotochromogens – develop pigment in the dark or light

Non-photochromogens -

M.avium-intracellulare complex (MAC or MAI) o In patients without AIDS:  Pulmonary infections in patients with preexisting pulmonary disease; cervical lymphadenitis; disseminated disease in immunocompromised, HIVnegative patients o In patients with AIDS:  Disseminated disease; environmental sources are natural water

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M. ulcerans o Indolent cutaneous and subcutaneous infections o Infections occur in tropical or temperate climates o Has not been isolated from the environment

Scotochromogens – pigmented in the dark © and does not intensify after exposure to light (D)

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Non-Photochromogens – nonpigmented regardless of grown in the dark or light

Different colony morphology seen on culture of one strain of M. avium complex

Nonphotochromogens – non-pigmented when grown in the dark (E) and after light exposure (F)

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Rapid-growers – colonies of NTM that appear on solid media in less than 7 days

Photochromogens M. kansasii o Chronic pulmonary disease; extra-pulmonary diseases (cervical lymphadenitis & cutaneous disease) o 3% of clinical illness known as TB o cross-reactive to PPD

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Rapid Growers -

M. abscesus o Disseminated disease in immunocompromised patients, skin and soft tissue infections, pulmonary infections, postoperative infections

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M. fortuitum o postoperative infections infections in breast augmentation andmedian sternotomy; skin and soft tissue infections

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M. chelonae o Skin and soft tissue infections, postoperative wound infections, keratitis

sensitive to standard antituberculous drugs such as rifampin habitat is tap water

M. marium o Cutaneous disease o Natural reservoir is fresh water and saltwater as a result of contamination from infected fish and other marine life M. kansasii colonies exposed to light

Smooth, multilobated colonies of M. fortuitum on Lowenstein-Jenssen medium

Fite-Faraco stain: single AFB in a nerve of patient with indeterminate leprosy

Borderline leprosy: Lesions are inflamed and succulent with central clearing, producing a “punched out” appearance, or may appear as plaques or bands with peripheral edges fading into normal surrounding skin. Central sensory deficits if present.

Mycobacterium Leprae H & E stain: Epithelioid granuloma with few scattered lymphocytes and a clear subepidermal zone

Borderline leprosy: lesions are multiform, from borderline tuberculoid appearance to more infiltrated nodules and plaques, with some loss of sensation at the center.

Classification of Leprosy -

clinical significance: o chronic granulomatous condition of peripheral nerves and mucocutaneous tissues, particularly the nasal mucosa

Tuberculoid Leprosy

Progression

Leptromatous leprosy

Of disease -

BL: thick erythematous plaques on the nose , right cheek and chin with some lesions appearing nodular.

laboratory identification o cannot be cultured on artificial media o can be grown in the footpads of mice and armadillo o acid-fast bacilli from nasal mucosa and other infeted areas in lepromatous leprosy o histopath and clinical findings in tuberculoid leprosy Indeterminate leprosy: on the extensor suface of extremities. Lesion is single, faintly hypochromic macule with illdefined borders; slightly erythematous with hypoesthetic areas in some parts. At times sensation is intact

Borderline leprosy: Uniformly and symmetrically distributed, infiltrated maculopapular lesions. No sensory impairment.

Borderline leprosy: In FiteFaraco stain AFB is present in moderate numbers seen within a nerve

Indeterminate leprosy in children: solitary, ill-defined, Hypopigmented macules, partially anesthetic. May progress to either tuberculoid or lepromatous forms. Tuberculoid leprosy: small ,single, circinate lesion with pink, elevated, finely granular,well-defined border. Central hypopigmented macule was insensitive to touch & pain. Associated with enlarged peripheral nerves.

Tuberculoid leprosy: H&E stain of an epithelioid granuloma with thick zone of lymphocytes destroying a nerve which is unrecognizable

Far advanced nodular lepromatous leprosy. Diffuse infiltration with nodules over the eyebrows, cheeks, ear lobes, nose, and chin.

Arm of patient with lepromatous leprosy: multiple typical nodules skin. Central sensory deficits if present.

Lepromatous leprosy: Diffuse infiltration, madarosis, and loss of eyelashes(diffuse lepromatosis)

H & E stain of skin section with active Lepromatous leprosy: highly vacuolated foam cells, normal appearing nerves, histiocytic granuloma. Fite-Faraco stain in Lepromatous leprosy: enormous numbers of AFB, in huge clumps, termed globi.

Treatment and prevention sulfones: Dapsone Rifampin Clofozamine For erythema nodosum leprosum: o Thalidomide o TNF-α inhibitor

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