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) plasma- associated 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

• (tb) – M. tuberculosis – M. bovis – M. africanum

• – 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 tuberculosis IncidenceTBC incidencia of TB in Magyarországon Hungary (újonnan(newly registered regisztrált, cases, aktív active betegek) 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

5000

4000 20%ooo

3000

2000 No. of cases of No. 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 !! spleen • fibrin envelope (protection)  reactivation in years – Dissemination by macrophages:

• miliary tb (blood) kidney • meningitis basilaris • kidneys, bones, spleen, bone marrow, intestine 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 – taken at early morning, pre-treated with 2% NaOH • Major media: – Löwenstein-Jensen (L-J), Sula, Dubos • Recommendation: 2 solid, 1 liquid • Solid media: – 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 Dubos Löwenstein-Jensen 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 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 ) lepromin – CD4+ cell dominance CD8+ cell dominance no or few bacteria in skin biopsy many bacteria in skin biopsy (+ also in blood) • formation • infiltrated nerves • hypo pigmented, insensitive skin • papular, nodular skin lesions lesions • amputation of nose, fingers • thickened peripheral nerves • facies leonina (lion face) • amputation of fingers, hands, feet • 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 , 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, 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