Mycobacteria Nocardia Actinomyces

Institute of Medical Microbiology Semmelweis University

Prof. Károly NAGY

NAGY KÁROLY MYCOBACTERIA (> 50 species, saprophitic) © aerobic, rod shape(0.4x3 µm), no spores or cilias © Can not be stained by Gram , once stained, reisist decoloration by acid or alcohol Acid-fast bacteria © Stained by Ziehl-Neelsen acid-fast dye © high level resistance towards chemicals Pathogenicity Species Reservoire Disease

Pathogenic M.tuberculosis human tbc M.leprae human lepra M.bovis bovine, human intestinal-,miliary tbc

Potentially M.avium- soil, birds, water disseminated and lung tbc Pathogenic intracellulare bovine (frequent in AIDS ) M.kansasii water, bovine lung tbc M.marinum fish, water sc.nodules, skin ulcers M.scrofulaceum soil, water granuloma, cervical ly.ad M.ulcerans human, environment sc.nodules, skin ulcers M.fortuitum-chelonei soil, water, animals sc.abscess,disseminated Ziehl-Neelsen stain M. tbc

„Cord-formation”

Gram negatíve Gram positive Complex (acid-fast )

acyl lipids

Lipid+LPS Mycolic acid porins (mycolyltrehalose: „cord-factor”)

D-arabinose D-galactose peptidoglycan lipid bilayer plasma memrane Mycobacterium tuberculosis

Culture: aerobic, slow growth : cell division 18 h

Sula (semisynthetic, Malachit-green)

Growth: clamps, bigger volumes Mycobacterium tuberculosis Pathogenesis Tbc develops in < 5% of those infected

Source of infection: Sick human animal alveols granuloma lympnodes Infection caverna Aerosol droplets dust per os (milk) contacts

Lung granuloma Mycobacterium tuberculosis Pathogenesis

alveolar macrophag

Surviving in phagolysosomes

2-4 weeks post infection: ‹CD4 T-cell cytokins activate macrophages

‹‹‹Granuloma and caverna formation: cytokine toxycity, complement activation, ischemia, macrophag enzym activity,etc.

High number of bacteria in sputum: primer and secunder infection, high resistance towards drying and chemicals ! Mycobacterium tuberculosis: Pathogenesis

Focal,cheesy necrosis: Caverna formation Mycobacterium tuberculosis (tbc): Clinical picture

Pulmonal: lung (middle/lower lobes), lymphnodes, pleura tünetek: weakness, fatigue,weight loss, caughing, spitting: yellow/green mucus, blood in the sputum, short breath, night sweating, pain in chest, back

In those infected disease develops <2years: 5%, > 2 years : additional 5-10%

• clinical diagnosis • x - ray • microscopy • culture Mycobacterium tuberculosis (tbc): Clinical picture

Extrapulmonal : haematogen or urogen dissemination, contact infectio

• gastrointestional (infected milk) • genito-urinary system (kidney tbc) • bone (deformity) • skin tbc (erythema multiforme, aquarium disease) • scrofula (neck swelling, ulcer) IMMUNITY

Gate of entrance Source of infection

PRIMER

SECUNDER M. tuberculosis: surviving strategies in macrophages

- high resistency of lipid/wax cell wall - neutralisation of free radicals - ammonia formation, which inhibits * formation of phagolysosomes * activity of lysosomal enzymes

„dormant” state M. tuberculosis INDUCED IMMUNE DESTRUCTION

I. type II.type III. type IV.type hypersensitivity anaphylaxias cytotoxikus immunkomplex delayed, tuberculin type IgE IgG IgG - - Complement Complement - --- T lymphocyte pollen,animal Rh, bact.,virus, intracellular fur, penicillin gyógyszer fungi, etc, microbe animal sera animal sera local,gene- self cell Co activation granuloma ralised, ADCC formation odema, his- lysis trombocyte tamin activation skinprobe - - skinprobe Tuberculin test: Mantoux - probe

Suitable to detect tbc infection or validation of the effect of BCG vaccination Purified,standardised tuberculoprotein : M.tbc PPD (Purified Protein Derivate)

5 TU intracutan inoculation, < 10 mm induration: negatív readafter48 h 10-20 mm induration: BCG-hypersensistivity > 20 mm induration: infekctious hypersensitivity Not M. tbc specifc Conversion (pozitív): M.tbc infection, BCG vaccination Reversion (negatív): early infection, miliary tbc, meningitis, AIDS, immunosuppression, morbilli,varicella, wooping cough , influenza, helmintiosis, old age THE HISTORY OF TUBERCULOSIS

•Kr.e. 1000 egyptian mummies – bone destrucion, abscess •Hyppokrates: aerosol, droplets infection •Middle ages: „phtysis”, the disease of the lung •Franz de la Boë (1614 – 1672) : „tuberculum” •London, XIX.c.:every 3. death due to tbc •Johan Lukas Schönlein (1793 – 1864): the clinical definition of tuberculosis (1832) •1882: Robert Koch: a ‘Koch-bacillus” the etiological factor 1890 – tuberculin, 1930- diagnostical meaning •1906: Albert Calmette és Camille Guerin : a „BCG”, the possibility of active immunization (1921) •1930: a Lübeck catastrophy: the significance of virulent/ avirulens strains, not interchangeable •1930: introduction of tbc screening programs,epidemy decrease •1945: WW II: epidemy increase •1950-1980: significant decreasing •1992: WHO Tbc: global problem •2000: WHO AIDS – Malaria – TBC GLOBAL EMERGENCY

Famous people who have had tuberculosis : Calvin Anton Chehov Branwell Bronté Emily Bronté Frédéric Chopin Descartes John Keats D.H. Lawrence Vivien Leigh Nelson Mandela Moliere George Orwell Paganini Edgar Allan Poe Richelieu cardinal Schiller Jean J. Rousseau Sir Walter Scott P.B.Shelly Spinoza R.L. Stevenson Desmond Tutu Simonetta Vespucci Margaret Gardner (a former Ms. World) EPIDEMIOLOGY : TB Incidence 2001

per 100 000 pop < 10 10 - 24 25 - 49 50 - 99 100 - 299 > 300 Tbc prevalencia - Hungary RISK FACTORS Tbc split by gender and age

MALES FEMALS

HOMLESS DIRCT CONTACT

IMMIGRANT

LONELY „Frigyes Korányi” National TBC and Pulmonological Institute INCIDENCE OF TUBERCULOSIS • TB is responsible for 25% of adult deaths in the developing world- more than those caused by diarrhea, malaria and AIDS combined • South Africa has one of the highest incidences: >200/100 000 • 35 people die of TB every day in South Africa • The incidence in the 'first world' is less than 10/100 000

THE GLOBAL EMERGENCY 1. 8-12 million new infections with M. tuberculosis per year 2. 2-3 million people die from TB per year 3. If present trends continue, deaths will exceed 4 million by 2004 4. There is also the emergence of multi-drug resistant M. tuberculosis 5. These trends, and the association of TB with HIV infection, has led to TB being declared A GLOBAL EMERGENCY by the World Health Organization (1993)

Laboratory diagnosis

The basics of the effective treatment of tbc: early, rapid and accurate diagnosis

Microscopy od stained smear Isolation, culture

10 4 bacterium/ml sensitivity 10 2 bacterium/ml low sensitivity long culture time: 4-8 hét Cytocentrifuge (Sauton – and Dubos media ) Laboratory diagnosis

BacTec automats : radioactive palmitinic acid/CO2 fluorescent detection turbidity other color reaction Rapid growth detection 4-14 nap

Biosensors:

< 1 h

Molecular detection : PCR, LCR RFPL, DNA typing, DNS-chip Drug sensitivity/ resistance determination ( hours, 1-2 days) Atypical and apathogenic mycobacteria

M. avium-intracellulare, M. kansasi , M. scrofulaceum M. smegmatis, M. phlei, stb induce mycobacteriosises characteristics : occurrence in the nature faster growth pigment formation niacin negativity different drug resistancy member of the normal flora disease: local (skin, lung, lymphnodes, etc.) processes no transmission from human to human, nozokomial infections generalized in AIDS THERAPY

• Stop infectivity • Inhibit drug resistance • Prevent remission of infection • Goal : complete recovery

Combination treatment, long time (6 -12 month) treatment Initial, intense phase: 3 -4 anti tuberculotics against extracellular bacteria Stabilisation phase: fewer drugs against intracellular, slow growing bacteria

INH (isonikotinic acid hidrazid ) - Rifampicin - Pirazinamid Ethambutol (AIDS, extrapulmonaltbc, meningitis ) Ciprofloxacin,ethionamid,cycloserin (Multiresistant M.tbc ) Clarithromycin,rifampicin,etambutol,clofazimin (M. avium-intracellulare ) Doxicyclin ( M. fortuitum-chelonei )

Multiresistant strains ! TBC – AIDS coinfections PREVENTION

1. Prevention of infection: General hygienic rules Effective treatment of those infected Eradication of infected cattles

2. Vaccina : BCG inoculation M.bovis live, attenuated strain BCG ( Bacillus Calmette-Guerin) 2 - 42 day after birth, partial resistency

3. Chemoprofilaxis :

INH monotherapy , Rifampicin (min. 6 month) Induces lepra (Hansen disease) , does not grow in media or cell culture, Only in mouse sole and in armadillo. Aerob, pálcika alakú, saválló baktérium : Ziehl – Neelsen festés Laboratory diagnosis : from skin disorders, nose mucosa Syphilis serological reactions are fals posistive in lepra The bacteria grow intracellularly in skin histiocytes, endothel cells and superficial nerves (Schwann-cells) . Optimal temperature: 30 C°

Tuberculoid lepra: normal cellular immunity, few bacteria growing, giant cells in granulomas Lepromin skin tests: positive

Lepromatic lepra: immune response towards M.leprae is weak skin, mucosa : large number of bacteria (histiocytes) Lepromin skin test: negative Immune response to only M.leprae is defective, cellular and humoral immune response towards other microorganisms are intact Antibodies are not protective Long incubation time: 10-15 year ! Gradual disease formation

Macular skin disorders: Insensibility ulcer Nodular skin disorders: „ lion face” (facies leontina) Fingers, extremities exposed to amputation because trophical Disorders, resorption of bones resulting in loss of nose cartilags or digits

Dapson (sulfonamid residue) Rifampin (inhibitor of bacterial RNS synthesis Clofazimin

Boswelia carteri (Tömjénfa) „Salai guggul” Nocardia genus

Obligate aerob, Gram positive, moderate acid fast , filamental bacteria

Occurrence : widespread, soil, water Domestic animals, dogs, cows (natural hosts) In Humans: N.asteroides N. braziliensis

Nocardiosis: chronic, lobular pneumonia, miliary tbc-like fever, weigh loss, chest pain. Lung abscess , haematogen dissemination:central nerve system abscesses Mycetoma : abscess in hand, leg, s.cutan connecting tissues

Laboratory diagnosis : sputum, pus, liquor smears Gram- and acid fast staining

Treatment: Surgical removal trimetoprim sulfonamids Actinomyces

Anaerobic, no spores, Gram positive, irregular shape rods Actinomyces israelii: non obligate anaerobic, slow growth (7-10 days) Actinomycosis

Pathogenesis : 2 -3 mm, yellow granules (mycelia) consisting of the bacterial bodies

Gram positive

Gram negative

Actinomyces mycelium Actinomycosis Pathogenesis : typical endogenous infection. Caries teeth, pharyngeal lavage, intestines, Vaginal flora Pathogenic process is initiated by : trauma, pyogenic infections, i.u. contraceptive devices Pelvic inflammation ABSCESS FISTULA DRAINING OF PUS

SKIN actinomycosis Lung actinomycosis Therapy: • Surgical removal • Penicillin • sulfonamids