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Basics of Mycobacterium spp for student.pptx
- 1. Basics of Mycobacterium spp SIW Dept of Clin Microbiology Updated: March 2023
- 2. Terminology • Mycobacteria are rod-shaped, aerobic bacteria that do not form spores. Although they do not stain readily, after being stained, they resist decolorization by acid or alcohol • Has mycolic acid that makes the cell wall gain hydrophobic attribute • Genus Mycobacterium are recognized 71 species and produced a spectrum of infections in humans and animals
- 3. Taxonomy ⚫Ordo : Actinomycetales ⚫Family : Mycobacteriaceae ⚫Genus : Mycobacterium ⚫Species : M. tuberculosis M. bovis M. africanum M. avium-intracellulare M. marinum M. kansasii M. fortuitum M. scrofulaceum M. leprae
- 4. Overview • First found in 1882 by Robert Koch • Appears as thin straight rods; but may appear as coccoid or filamentous in artificial media. In size the cell 0.4x 3.0µm • Doesn’t form spore • Non motile • Does not own capsule • The cell wall is complex containing large lipid, composed of fraction A,B,C,D
- 5. Cell structure The Component of cell wall bacilli : Lipids (Fraction :wax A,B,C,D ) Protein Polysaccharides
- 6. Cell structure The Component of cell wall bacilli : Lipids (Fraction :wax A,B,C,D ) Protein Polysaccharides
- 8. Clinical manifestation • Pulmonary tuberculosis • 85% of all cases • Extrapulmonary tuberculosis • Ie. Skin TB, scrofuloderma, etc
- 9. Diagnosis Microscopy • Ziehl Neelsen • Kinyoun • Truant flurochrom Culture • Egg based media (Lowenstein Jensen) • Agar based media (Middlebrook 7H10) • Broth based media (Middlebrook 7H9) Nucleic Acid Base Test • Rapid molecular test • PCR Immunodiagnosis • Tuberculin test • Interferon gamma release assay (IGRA)
- 10. Culture • Optimum growth 35 – 37 oC; better in 5 – 10% CO2 • Incubation can up to 4 – 8 weeks
- 11. The long life span • Comparing the lifespan and the proliferative potential of E. coli and MTB, we see a reverse relationship. E. coli live short, and grow fast. It just takes 18-20 minutes for them to duplicate. Conversely, MTBs live long, are quite tolerant to different environments, and grow so slowly that their duplication time exceeds 18 hrs. • The duplication time of Mycobacterium leprae is even longer, so that all the cultivation efforts have failed. Although nobody knows exactly how long these bacteria can live since they cannot be cultivated, it can be postulated that they live very long. Wang, et al., J Clin Exp Pathol 2014, 4:3 DOI: 10.4172/2161-0681.100017
- 12. The long life span Mycobacteria Non Mycobacteria Slow response to everything, duplication is very long Relatively fast response, duplication is relatively fast Environmental changes Environmental changes Does not have major effect 🡪 pathogenicity is retained May have major effect 🡪 pathogenicity is short-lived
- 13. Runyon Classification • Runyon classified acid fast bacilli by their growth speed
- 15. Taxonomy Domain: Bacteria Phylum: Actinomycetota Class: Actinomycetia Order: Mycobacteriales Family: Mycobacteriaceae Genus: Mycobacterium Species: M. tuberculosis
- 16. Overview • M. tuberculosis is an intracellular pathogen that is able to establish lifelong infection • May dormant for a very long time • Human is the only host • In contrast with most phagocytized bacteria, M. tuberculosis prevents fusion of the phagosome with lysosomes (by blocking the specific bridging molecule, early endosomal autoantigen 1 (EEA1) and PI3P at late maturation • Phagosome is then fuse with other intracellular vesicles, permitting access to nutrients and facilitating intravacuole replication
- 17. Virulence factors Lipids • Mycolic acids (long-chain fatty acids C78–C90) • Muramyl dipeptide (from peptidoglycan) complexed with mycolic acids can cause granuloma formation • Phospholipids induce caseous necrosis • Serpentine cords (“cord factor” (trehalose-6,6′- dimycolate)) 🡪 inhibits migration of leukocytes, causes chronic granulomas, and can serve as an immunologic “adjuvant.” Protein • Tuberculin • And many other proteins Polysaccharides • Induce immediate hypersensitivity • Acts as antigen
- 18. Virulence factor • Serpentine cord / trehalose dimycolate • Glycolipid molecule in the cell wall • The primary lipid in M tb wall • Its presence gives M tb the slender appearance • Induce immune system, including granuloma formation • Catalase enzyme • Withstand many antibacterial agents M tb does not produce toxin
- 19. Interfering in phagolysosome biogenesis • Living Mycobacterium tuberculosis persists in macrophage phagosomes by interfering with phagolysosome biogenesis • Living M. tuberculosis secretes a lipid phosphatase, SapM, that hydrolyzes PI3P, inhibits phagosome–late endosome fusion in vitro, and contributes to inhibition of phagosomal maturation • PI3P executes various steps in membrane trafficking, endosomal protein sorting, and multisubunit enzyme assembly at the membrane, including phagosomal maturation Proc Natl Acad Sci U S A. 2005 Mar 15; 102(11): 4033–4038
- 20. Interfering in phagolysosome biogenesis X Engulfing the bacilli Lysosome moves near M.tb prevents biogenesis Other endosome moves near Fusion between phagosome and endosome M.tb survives intracellular
- 21. Evading immune system • Phagocytized bacteria are able to evade macrophage killing mediated by reactive nitrogen intermediates formed between nitric oxide and superoxide anions by catabolizing the oxidants that are formed, thus replication is possible while staying intracellullarly
- 22. Evading immune system • In response to infection with M. tuberculosis, macrophages secrete interleukin (IL)-12 and tumor necrosis factor (TNF)-α. increase localized inlammation with the recruitment of T cells and natural killer (NK) cells into the area of the infected macrophages, inducing T-cell differentiation into TH1 cells (T-helper cells), with subsequent secretion of interferon (IFN)-γ. • In the presence of IFN-γ, the infected macrophages are activated, leading to increased phagosome-lysosome fusion and intracellular killing. In addition, TNF-α stimulates production of nitric oxide and related reactive nitrogen intermediates, leading to enhanced intracellular killing.
- 23. Inducing Apoptosis • Mtb strain H37Ra induces bystander apoptosis in uninfected macrophages in a dose-and time-dependent manner, and significant apoptosis is seen 48 h after H37Ra infection (link) • Many mycobacteria induces apoptosis, but Mtb infected macrophages have also been shown to inhibit the extrinsic apoptosis pathway by modifying the expression of death receptors such as Fas (CD95) and the soluble TNF receptor 2 (sTNFR2)(link) (link) whil e
- 25. Illness progress vs diagnosis
- 26. Tuberculin test Positive PPD test indicates: • Person with immunity • Person infected with M tb (past history or present) • Person who has been vaccinated • Person with other mycobacteria infection (false positive) Negative PPD test indicates: • Person with no immunity • Person with no history of contact with M tb • Person with anergy (false negative)
- 27. IGRA Interferon-Gamma Release Assays (IGRAs) are whole-blood tests that can aid in diagnosing Mycobacterium tuberculosis infection; does not differentiate between latent TB and active TB Two FDA-approved tests: • QuantiFERON®-TB Gold In-Tube test (QFT-GIT); • T-SPOT®.TB test (T-Spot) (link)
- 28. IGRA
- 29. IGRA
- 30. IGRA
- 31. Specimen collection • Depends on location of infection • Mostly sputum from individual with pulmonary tb • Other specimen can also be taken (refer to CMPH to prepare many other specimens, including tissue specimen)
- 32. Basic Interpretation of ZN Smear link here
- 33. TB Management in Indonesia • Peraturan Presiden Nomor 67 tahun 2021 tentang Penanggulangan Tuberkulosis (link) • Pedoman Nasional Pelayanan Kedokteran Tatalaksana Tuberkulosis (link) • International Standards for TB Care (ISTC) 3rd Version (link)
- 36. Thank You