Mycobacterium tuberculosis

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MYCOBACTERIUM

Mycobacteria - in general • Aerobic rods • Special cell wall composition (rich in lipids) – no Gram staining – hydrophobic cell surface  very resistant!

• Very slow growth! – generation time: 12-24 hrs (versus E. coli: 30 mins) – colony formation: up to 8 weeks !! (M. leprae can not be cultured at all)

Cell wall structure of Mycobacteria

VERY RICH IN LIPIDS !!

Mycobacterial cell wall structure. The components include the (A) plasma membrane, (B) peptidoglycans, (C) arabinogalactan, (D) mannose-capped lipoarabinomannan, (E) plasmaassociated and cell wall-associated proteins, (F) mycolic acids, and (G) glycolipid surface molecules associated with the mycolic acids. (Redrawn from Karakousis et al: Cell Microbiol 6:105-116, 2004.)

Mycobacteria - I obligate pathogens • Tuberculosis (tb) – M. tuberculosis – M. bovis – M. africanum

• Leprosy – M. leprae

Mycobacteria - II Atypical Mycobacteria • facultative pathogens or apathogens • disease = “mycobacteriosis”       

M. kansasii M. avium-intracellulare M. ulcerans M. marinum M. scrofulaceum M. fortuitum-chelonei M. smegmatis

Mycobacterium tuberculosis

Incidence of TB inMagyarországon Hungary TBC incidencia (újonnan regisztrált, aktív betegek) (newly registered cases, active patients) 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 1950 1955 1960 1965 1970 1980 1985 1990 1995 2000 2005

TB incidence since 1990

No. of cases

5000 4000

20%ooo

3000 2000

1000 0

év

Tuberculosis surveillance programme Korányi Bulletin, 2013. 1. szám

(2012)

Pathogenesis • Entry: aerosol (dust), bovine milk • Lungs: multiplication within alveolar macrophages (IC!) - localisation • Possibilities: – activated macrophages (CD4+ T cells – cytokin production) eliminate bacteria – if too many bacteria: necrotized loci (caverna formation) • BACTERIA IN SPUTUM !! • fibrin envelope (protection)  reactivation in years

spleen

– Dissemination by macrophages: • miliary tb (blood) • meningitis basilaris • kidneys, bones, spleen, bone marrow, intestine

kidney

Clinical features • Symptoms: – – – –

cough (sputum) night sweats malaise, weakness weight loss

• usually localised to lungs in immunocompetent persons

TB and HIV • cellular immunity is defected • progression to active disease within 1 year of exposure: 10% • appears usually first before other infections • more often extrapulmonary forms • more often severe forms (death)

Tuberculosis in the Vác mummies • Vác, Dominican church • 200-year old, naturally mummified bodies (1731-1838) • Clear documentation • CT scan • Detection of bacterial DNA by PCR (bones and tissue!) • 55% had tuberculosis Fletcher et al: Widespread occurrence of Mycobacterium tuberculosis DNA from 18th–19th century Hungarians. Am J Phys Antrop, 2003, 120:144-152.

TB diagnostics 1. Direct microscopic examination • •

• •

Directly from the sample or from centifuged sediment Pre-treatment of non-sterile samples! (NALC*-NaOH) Atypical mycobacteria present in tap water !! (e.g. M. gordonii) Ziehl-Neelsen staining or fluorochrom (rodmamin, auramin)

*NALC = N-acetyl-L-cystein

Ziehl-Neelsen staining “acid fast staining” • Carbol-fuchsin (on filter paper!) – heating 3x (until steaming)

• Differentiation: acidic alcohol – 3% HCl, 96% ethanol – “acid fast”

• thorough water washing • counter staining 1-2’ – methylene blue (or malachite green)

• careful rinsing, drying with filter paper

Acid fast preparations

Cord formation (from liquid culture, characteristic for virulent strains) (6,6’-dimycolil-trehalose)

TB diagnostics 2. Cultivation •

Specimen: mostly sputum –



Major media: –

• •

Löwenstein-Jensen (L-J), Sula, Dubos

Recommendation: 2 solid, 1 liquid Solid media: – – –



taken at early morning, pre-treated with 2% NaOH

4-8 weeks L-J, Ogawa, Stonebrink (= egg based) Agar based

Liquid media: – –

3-21 days (early detection methods – e.g. radioactive carbon source) Bactec 12B, MGIT, BioFM, etc.

Culture media

Sula Löwenstein-Jensen

Dubos

Bacteriological status of new cases in 2007 Only culture

smear

TB diagnostics 3. Molecular biological methods • Aim: direct detection, resistance determination, typing • ALWAYS ALONG WITH CULTURE !! • Nucleic acid amplification techniques (NAT) – False + : contamination, also dead bacteria – False - : small amount, inhomogeneous distribution in the sample, inhibitors

• Hybridization • RFLP • Sequencing

Tuberculin test (Mantoux-probe) • PPD (purified protein derivative), intracutan • Cellular immunity – delayed type allergy • Evaluation in 48 hrs: – < 10 mm: negative – 10-20 mm: BCG vaccination  – > 20 mm: infection

• Negativity may occur: – immunosupression – severe froms (disseminated tb, meningitis) – recent viral infection (vaccination!)

TB treatment • First line drugs: – INH, Streptomycin, Rifampin, Ethambutol

• Second line drugs: – FQ (ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin), cycloserin, PAS, Ethionamid, parenteral amynoglycosides (kanamycin, capreomycin, amikacin), even Augmentin (!)

TB resistance • MDR: – INHR, rifampicinR – treatable with second line drugs – ~ 5% in Hungary

• XDR: – INHR, rifampicinR , FQR + R to minimum 1 parenteral aminoglycoside

• XXDR: – Resistant to all! – A few strains also in Hungary!

Prevention 1. BCG vaccination • BCG: Bacillus Calmette-Guerin • Live, attenuated M. bovis • Given at the hospital (first 6 weeks of life) 2. Chemoprophylaxis • Direct contacts of actively ill patients • INH or rifampin, for a long time

Mycobacterium leprae

Prevalence of leprosy worldwide Now: • cca. 0,5 million cases / year • mostly in India, Brazil

Characteristics of M. leprae • acid fast rod, cigar-like • NOT culturable on media ! – only: sole of mouse, armadillo

• temperature optimum: 30oC – skin, peripheral nerves

• intracellular (endothel, Schwann-cells) • clinical forms: – tuberculoid …. lepromatous

Types of leprosy Tuberculoid

Lepromatous

benign form

malignant form

hardly infectious

very infectious

lepromin + (cell-mediated immunity ) CD4+ cell dominance

lepromin – CD8+ cell dominance

no or few bacteria in skin biopsy

many bacteria in skin biopsy (+ also in blood)

• granuloma formation • hypo pigmented, insensitive skin lesions • thickened peripheral nerves • amputation of fingers, hands, feet

• • • •

infiltrated nerves papular, nodular skin lesions amputation of nose, fingers facies leonina (lion face)

• sharp edged erythemic ring lesions (= erythema nodusum leprosum) asymmetric nerve involvement

symmetric nerve involvement

Acid-fast stains of skin biopsies from patients with (A) tuberculoid leprosy, (B) borderline tuberculoid leprosy, (C) borderline lepromatous leprosy, and (D) lepromatous leprosy. Note that there is a progressive increase in bacteria going from the tuberculoid form to the lepromatous form of the disease.

Tuberculoid form

Erythema nodusum leprosum

Lepromatous form

Facies leonina

What is the transmission of leprosy? • In the lepromatous form it is present in nasal exudates and skin lesions – Direct (long-term) contact (wound exudate) – Inhalations (infectious aerosol)

• Tuberculoid form: low infectivity • Other routes ?? – Mother’s milk – Insect vectors

Treatment of leprosy • Tuberculoid: rifampin + dapsone (1/2 year) • Lepromatous: + clofazimine (1 year) • separation of patients • Vaccination? – under development (many years to go…) – BCG ?

Atypical Mycobacteria Runyon classification I-IV. Runyon –I. Slow-growing, photochromogenic • M. kansasii – Source: water, cows – Disease: lung-tb (esp. AIDS patients!)

• M. marinum – Sweet and salt water, fishes – Skin lesion, subcutan nodules

Atypical Mycobacteria Runyon classification I-IV. Runyon –II. Slow-growing, scotochromogenic • M. scrofulaceum – – – – –

soil, water granulomatous cervical lymphadenitis entrance: oropharynx usually children surgical removal

Atypical Mycobacteria Runyon classification I-IV. Runyon –III. Slow-growing, non-chromogenic • M. avium-intracellulare – soil, water, animals – lung- and disseminated tb – immuncompromised (AIDS!) patients

• M. ulcerans – soil, water – skin lesion, abscesses

Atypical Mycobacteria Runyon classification I-IV. Runyon –IV. Rapid-growing (<7 days), non-chromogenic

• M. fortuitum-chelonei • M. abscessus – soil, water, animals – trauma  subcutaneous tissues – rarely cause disseminated infections

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Mycobacterium tuberculosis

MYCOBACTERIUM Mycobacteria - in general • Aerobic rods • Special cell wall composition (rich in lipids) – no Gram staining – hydrophobic cell surfac...

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