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Tuberculose
Dr. Svetoslav Dimitrov Todorov
Ana Netrebko as Violetta in La Traviata by Giuseppe Fortunino Francesco Verdi (10 October 1813 – 27 January 1901)
Mycobacterium is a genus of over 190 species in the
phylum Actinomycetota, assigned its own
family, Mycobacteriaceae. This genus includes pathogens known
to cause serious diseases in mammals, including tuberculosis (M.
tuberculosis) and leprosy (M. leprae) in humans.
The Greek prefix myco- means 'fungus', alluding to this
genus' mold-like colony surfaces. Since this genus has cell
walls with a waxy lipid-rich outer layer that contains high
concentrations of mycolic acid, acid-fast staining
is used to emphasize their resistance to acids,
compared to other cell types.
Mycobacterial species are generally aerobic,
non-motile, and capable of growing with minimal
nutrients. The genus is divided based on each species’
pigment production and growth rate.
While most Mycobacterium species are
non-pathogenic, the genus' characteristic complex cell wall
contributes to evasion from host defenses.
History
Mycobacteria have historically been categorized through phenotypic testing, such as the Runyon
classification of analyzing growth rate and production of yellow/orange carotenoid pigments.
Group I contains photochromogens (pigment production induced by light),
Group II comprises scotochromogens (constitutive pigment production),
and the non-chromogens of
Groups III and IV have a pale yellow/tan pigment, regardless of light exposure.
Group IV species are "rapidly-growing" mycobacteria compared to the "slowly-growing" Group III
species because samples grow into visible colonies in less than seven days.
Because the International Code of Nomenclature of Prokaryotes (ICNP) currently recognizes
195 Mycobacterium species, classification and identification systems now rely on DNA
sequencing and computational phylogenetics.
The major disease-causing groups are the
M. tuberculosis complex (tuberculosis),
M. avium complex (mycobacterium avium-intracellulare infection),
M. leprae and M. lepromatosis (leprosy (lepra, in Portugues)), and
M. abscessus (chronic lung infection).
Proposed division of the genus
Gupta et al. have proposed dividing Mycobacterium into
five genera, based on an analysis of 150 species in this
genus. Due to controversy over complicating clinical
diagnoses and treatment, all of the renamed species have
retained their original identity in the Mycobacterium genus
as a valid taxonomic synonym:
•Mycobacterium based on the Slowly-Growing
Tuberculosis-Simiae clade
•Mycobacteroides based on the Rapidly-Growing
Abscessus-Chelonae clade
•Mycolicibacillus based on the Slowly-Growing Triviale
clade
•Mycolicibacter based on the Slowly-Growing Terrae
clade
•Mycolicibacterium based on the Rapidly-Growing
Fortuitum-Vaccae clade
Morphology
① Mycobacteria are aerobic with 0.2-
0.6 µm wide and 1.0-10 µm long rod
shapes.
② They are generally non-motile, except
for the species Mycobacterium
marinum.
③ Mycobacteria possess capsules and
most do not form endospores.
④ The distinguishing characteristic of
all Mycobacterium species is a
thick, hydrophobic, and mycolic acid-
rich cell wall made
of peptidoglycan and arabinogalactan,
with these unique components
offering targets for new tuberculosis
drugs.
Scanning Electron Micrograph (SEM) image depicted a number
of Mycobacterium tuberculosis bacteria. Magnification: 21228x.
Physiology
① Many Mycobacterium species readily grow with minimal nutrients,
using ammonia and/or amino acids as nitrogen sources
and glycerol as a carbon source in the presence of mineral salts.
② Temperatures for optimal growth vary between species and media
conditions, ranging from 25-45 °C.
③ Most Mycobacterium species, including most clinically relevant species,
can be cultured in blood agar.
④ Some species grow very slowly due to extremely long reproductive
cycles, such as M. leprae requiring 12 days per division cycle compared
to 20 minutes for some E. coli strains.
Ecology
① Whereas Mycobacterium tuberculosis and M. leprae are pathogenic, most
mycobacteria do not cause disease unless they enter skin lesions of those
with pulmonary and/or immune dysfunction, despite being widespread
across aquatic and terrestrial environments.
② Through biofilm formation, cell wall resistance to chlorine, and association
with amoebas, mycobacteria can survive a variety of environmental
stressors.
③ The agar media used for most water testing does not support the growth of
mycobacteria, allowing it to go undetected in municipal and hospital
systems.
Protein-Coding Genomic Information
Number of Protein Coding GenesOrganism
5,289M. intracellulare
5,084M. colombiense
1,603M. leprae
3,995M. tuberculosis
6,602M. smegmatis
4,948M. chelonae
Genomics
Hundreds of Mycobacterium genomes have been completely sequenced.
The genome sizes of mycobacteria range from (e.g. in M. leprae) to quite
large ones, such as that as M. vulneris, encoding 6,653 proteins, larger than
the ~6000 proteins of eukaryotic yeast.
Pathogenicity
Mycobacterium tuberculosis complex
Mycobacterium tuberculosis can remain latent in human hosts for decades after an initial
infection, allowing it to continue infecting others. It has been estimated that a third of the world
population has latent tuberculosis (TB).
M. tuberculosis harbors many virulence factors, which can be divided across lipid and fatty
acid metabolism, cell envelope proteins, macrophage inhibitors, kinase proteins, proteases,
metal-transporter proteins, and gene expression regulators.
Several lineages such as M. tuberculosis var. bovis (bovine TB) were considered separate
species in the M. tuberculosis complex until they were finally merged into the main species in
2018.
Leprosy (lepra – in portugues)
The development of Leprosy is caused by infection with either Mycobacterium
leprae or Mycobacterium lepromatosis, two closely related bacteria. Roughly 200,000 new
cases of infection are reported each year, and 80% of new cases are reported in Brazil, India,
and Indonesia. M. leprae infection localizes within the skin macrophages and Schwann cells
found in peripheral nerve tissue.
Nontuberculosis Mycobacteria
 Non-tuberculosis Mycobacteria (NTM), which exclude M. tuberculosis, M. leprae, and M.
lepromatosis, can infect mammalian hosts.
 These bacteria are referred to as "atypical mycobacteria." Although person-to-person
transmission is rare, transmission of M. abscessus has been observed between patients
with cystic fibrosis.
 The four primary diseases observed in humans are chronic pulmonary disease,
disseminated disease in immunocompromised patients, skin and soft tissue infections, and
superficial lymphadenitis. 80-90% of recorded NTM infections manifest as pulmonary
diseases.
 M. abscessus is the most virulent rapidly-growing mycobacteria (RGM), as well as the
leading cause of RGM based pulmonary infections. Although it has been traditionally
viewed as an opportunistic pathogen like other NTMs, analysis of various virulence factors
(VFs) have shifted this view to that of a true pathogen. This is due to the presence of
known mycobacterial VFs and other non-mycobacterial VFs found in other prokaryotic
pathogens.
Virulence factors
① Mycobacteria have cell walls 
with peptidoglycan, arabinogalactan, 
and mycolic acid; a waxy outer mycomembrane 
of mycolic acid; and an 
outermost capsule of glucans and secreted 
proteins for virulence. 
② It constantly remodels these layers to survive in 
stressful environments and avoid host immune 
defenses. 
③ This cell wall structure results in colony surfaces 
resembling fungi, leading to the genus' use of the 
Greek prefix myco-. This unique structure 
makes penicillins ineffective, instead requiring a 
multi-drug antibiotic treatment of isoniazid to 
inhibit mycolic acid synthesis, rifampicin to 
interfere with transcription, ethambutol to hinder 
arabinogalactan synthesis, and pyrazinamide to 
impedeCoenzyme A synthesis.
Mycobacterial Infection Information
Reported Cases (Region, 
Year)Known TreatmentsCommon Symptoms of 
InfectionOrganism
1.6 Million (Global, 2021)isoniazid INH, rifampin, 
pyrazinamide, ethambutol.
Fatigue, weight loss, fever, 
hemoptysis, chest pain.M. tuberculosis
133,802 (Global, 2021)dapson, rifampicin, 
clofazimine.
Skin discoloration, nodule 
development, dry skin, loss 
of eyebrows and/or 
eyelashes, numbness, 
nosebleeds, paralysis, 
blindness, nerve pain.
M. lepraeM. lepromatosis
3000 (US, 
Annual estimated)
clarithromycin, 
azithromycin, amikacin, 
cefoxitin, imipenem.
Tender skin, development 
of boils or pus-filled 
vesicles, fevers, chills, 
muscle aches.
M. avium complex
Unknownclarithromycin, amikacin, 
cefoxitin, imipenem.
Coughing, hemoptysis, 
fever, cavitary lesions.M. abscessus complex
Diagnosis
The two most common methods for visualizing these acid-fast bacilli as bright red
against a blue background are the Ziehl-Neelsen stain and modified Kinyoun stain.
Fite's stain is used to color M. leprae cells as pink against a blue background. Rapid
Modified Auramine O Fluorescent staining has specific binding to slowly-growing
mycobacteria for yellow staining against a dark background.
Newer methods include Gomori-Methenamine Silver staining and Perioidic Acid
Schiff staining to color Mycobacterium avium complex (MAC) cells black and pink,
respectively.
While some mycobacteria can take up to eight weeks to grow visible colonies from a
cultured sample, most clinically relevant species will grow within the first four weeks,
allowing physicians to consider alternative causes if negative readings continue past
the first month.
The Ziehl-Neelsen stain, also known as the acid-fast stain, is a
bacteriological staining technique used in cytopathology and microbiology to
identify acid-fast bacteria under microscopy, particularly members of
the Mycobacterium genus. This staining method was initially introduced by Paul
Ehrlich (1854–1915) and subsequently modified by the German
bacteriologists Franz Ziehl (1859–1926) and Friedrich Neelsen (1854–1898)
during the late 19th century.
Basic steps of the Ziehl-Neelsen staining procedure
The acid-fast staining method, in conjunction with auramine phenol staining, serves as the
standard diagnostic tool and is widely accessible for rapidly diagnosing tuberculosis (caused
by Mycobacterium tuberculosis) and other diseases caused by atypical mycobacteria, such
as leprosy (caused by Mycobacterium leprae) and Mycobacterium avium-
intracellulare infection (caused by Mycobacterium avium complex) in samples like sputum, gastric
washing fluid, and bronchoalveolar lavage fluid. These acid-fast bacteria possess a waxy lipid-rich
outer layer that contains high concentrations of mycolic acid, rendering them resistant to
conventional staining techniques like the Gram stain.
After the Ziehl-Neelsen staining procedure using carbol fuchsin, acid-fast bacteria are observable
as vivid red or pink rods set against a blue or green background, depending on the
specific counterstain used, such as methylene blue or malachite green, respectively. Non-acid-fast
bacteria and other cellular structures will be colored by the counterstain, allowing for clear
differentiation.
Microscopic visualisation of the acid-fast bacteria Mycobacterium tuberculosis (left) and Mycobacterium 
leprae (right) and background cellular material in blue using the Ziehl–Neelsen stain
Mycobacterium tuberculosis Mycobacterium leprae
History of Tuberculosis
Tuberculosis is one of the ancients' diseases (with some evidences 
from cave man been infected)
Tuberculosis (TB) is the leading cause of death in the world in the 
group of bacterial infectious diseases. 
The TB affects more then 1.8 billion individuals per year, equal of 
1/3 of the entire world population.
Robert Koch discovered Mycobacterium tuberculosis in 1882. 
Heinrich Hermann Robert Koch
11 December 1843 – 27 May 1910) was a German physician and microbiologist. As the
discoverer of the specific causative agents of deadly infectious diseases
including tuberculosis, cholera and anthrax, he is regarded as one of the main founders of
modern bacteriology.
Löwenstein–Jensen medium, more commonly known as LJ medium, is a growth
medium specially used for culture of Mycobacterium species, notably Mycobacterium
tuberculosis.
When grown on LJ medium, M. tuberculosis appears as brown, granular colonies
(sometimes called "buff, rough and tough"). The medium must be incubated for a
significant length of time, usually four weeks, due to the slow doubling time of M.
tuberculosis (15–20 hours) compared with other bacteria.
The medium is named after the Austrian pathologist Ernst Löwenstein (1878–1950)
and the Danish medical doctor Kai Adolf Jensen (16.7.1894-2.5.1971).
Cultural characteristics:
A) Solid media:
Containing egg – Löwenstein–Jensen medium, Petragnin, Dorset’s egg
Containing blood – Tarshis medium
Containing potato – Pawlowsky’s medium
The usual composition of Löwenstein–Jensen medium as applicable 
to M. tuberculosis is: 
Malachite green; Glycerol; Asparagine; Potato starch; Coagulated eggs; 
Mineral salt solution; Potassium dihydrogen phosphate; Magnesium sulfate; 
Sodium citrate.
Low levels of penicillin and nalidixic acid are also present in LJ medium to 
inhibit growth of Gram-positive and Gram-negative bacteria, to limit growth 
to Mycobacterium species only. 
Presence of malachite green in the medium inhibits most other bacteria. 
Presence of glycerol enhances the growth of M. tuberculosis.
For cultivation of M. bovis, glycerol is omitted, and sodium pyruvate is added.
The medium appears green, opaque, and opalescent.
Cultural characteristics:
Cultural characteristics:
A) Liquid media:
Dubo’s, Middlebrooke’s, Prouskeur & Beck’s, Sula’s & Sauton’s.
Liquid media useful for sensitivity tests, for extraction of Ag (antigens) and for vaccines 
preparations
growth in liquid media – pellicle at surface
Dubo’s medium with Tween 80 – provide better diffuse growth
Virulent strain – Serpentine cords
Avirulent strain – Dispersed growth
Use of chick embryo & tissue cultures
Resistance of Mycobacterium tuberculosis
Not heat resistance
But resistant to chemical disinfectants such as:
 Phenol
 Destroyed by tincture iodine - 5 min contact time
 80% ethanol – 2-10 minites contact time
 Sensitivity to formaldehyde and glutaraldehyde 
Viability: 
Sputum (escarro): 20 - 30 hrs
Droplets: 8 – 10 days
Cultures: 6 – 8 months
Antigenic structure
Cell wall antigens:
 Peptidoglycan layer
 Arabinogalactan layer
 Mycolic acid layer
 Mycosides
Cytoplasmic Antigens
(Protein antigens)
Mycolic acid
 Difficult to stain
 Difficult to phagocytes
 Intracelular survival
 Hypersensitivity
 Slow growth
 Resistant to chemical disinfectants 
Active TB disease
Mycobacterium tuberculosis
 Awake and multiplying (Active vegetative cells)
 Cause damage to the lung 
Host (Homo sapiens) 
 Most often feels sick (all symptoms)
 Contagious (before pills started)
 Usually have a positive tuberculin skin test
 Chest X-ray is often abnormal (with pulmonary TB)
Primary tuberculosis:
Mostly asymptomatic
Some may have fly like symptoms; chest pain; mild fever and lack of appetite
Within 3 weeks, cell mediated immunity checks the M. tuberculosis
Engulfed M. tuberculosis in alveoli forms a lesion called Ghon focus in lower lobe 
(Anton Ghon, Austrian pathologist)
Some M. tuberculosis are transported to hilar lymph nodes. 
Ghon focus in lower lobe together with the enlarged hilar 
lymph nodes is called Primary Complex (Ghon Complex). 
Described by Karl Ernst Ranke, German pulmonologist. 
Secondary tuberculosis: (in 10% cases facilitated by)
 HIV infection
 Alcoholism and liver cirrhosis
 Malnutrition
 Diabetes
 Steroid and immunosuppressive therapy
 Old age
Secondary tuberculosis: (caused by reactivation(immunosuppression) of the primary lesion)
 Spreads to upper lobes
 Granuloma occurs in apex of lungs
 Memory T cells releases cytokines
 Causes tissue destruction and necrosis called 
tuberculomas (caeseous necrosis)
 Cavities may rupture into blood vessels, 
spreading M. tuberculosis through body
 Causing systematic Miliary tuberculosis.
Secondary tuberculosis:
Miliary tuberculosis may develop in any organs of the 
body
 Certain tissues like heart, striated muscles, thyroid 
and pancreas are resistant
 Localization sites are the bone marrow, eye, lymph 
nodes, liver, spleen, kidney, adrenal, prostate, seminal 
vesicles, fallopian tubes, endometrium and meninges.
Secondary tuberculosis:
Clinical signs:
 Temperature elevation 
usually in mid-
afternoon, night 
sweats, weakness, 
fatigability, loss of 
appetite and loss of 
weight.
 Productive cough, 
blood-streaked 
sputum (hemoptysis).
Secondary tuberculosis:
 Cervical lymph nodes – Scrofula
 Eye – Ocular tuberculosis
 Meninges – Tuberculous meningitis
 Kidney – Renal tuberculosis
 Tuberculosis in Adrenals – souses Addison’s disease
 Bones – Tuberculous osteomyelitis
 Fallopian tubes and epididymis – Genital tuberculosis 
Laboratory diagnosis:
 Specimen depending on 
clinical presentation –
sputum, pus, urine, CSF, 
pleural/ascitic fluid
 Pulmonary tuberculosis –
Early morning sputum sample 
on 3 consecutive days 
(Bacillary shedding is 
intermittent)
 Sputum is collected in wide 
mouth containers. 
Concentration methods: Petroff’s commonly used 
(This approach destroy other present bacteria)
 Sputum + equal volume of 4% NaOH
 Incubate at 37oC for 20 min with frequent shaking till clearing
 Centrifuge at 3000 xg for 30 min. Discard supernatant
 Neutralization by 10N HCl
 Deposit used for smear, culture, animal inoculation. 
Culture: Inoculate 2 slopes of Lowenstein Jensen (LJ) medium
 (Detects as few as 10-100 bacilli / ml) 2 tubes of medium
 Keep incubator at 37oC with 5-10% CO2
 Examine every day for growth for rate of culture identification.
summary
Tuberculosis
• Tuberculosis is caused by Mycobacterium tuberculosis (acid-fast strain).
• Transmitted from human to human 
• M. tuberculosis may be ingested by alveolar macrophages; if not killed, the bacteria 
reproduce in the macrophages.
• Lesions formed by M. tuberculosis are called tubercles; dead macrophages and bacteria 
form the caseous lesion that might calcify and appear in an X ray as a Ghon complex.
• Liquefaction of the caseous lesion results in a tuberculous cavity in which M. tuberculosis 
can grow.
• Chemotherapy usually involves 3~4 drugs 
(rifampin, INAH, pyrazinamide, ethambutol, SM) 
taken for several months; multidrug-resistant (MDR)
M. tuberculosis is becoming prevalent.
• 3 million death each year
• TB is the leading killer among the infectious 
• diseases
• A positive tuberculin skin test can indicate either an active case of TB, or prior infection, 
or vaccination and immunity to the disease.
• Mycobacterium bovis causes bovine tuberculosis and can be transmitted to humans by 
unpasteurized milk.
• M. bovis: <1% U.S. cases, not transmitted from human to human
• BCG vaccine for tuberculosis consists of a live, avirulent culture of M. bovis. Not widely 
used in U.S.
• M. avium-intracellulare complex (MAC) infects patients in the late stages of HIV 
infection.
Tuberculosis
Diagnosis: Tuberculin skin test screening
+ = current or previous infection
Followed by X-ray or CT, acid-fast staining of
sputum, culturing bacteria
Tuberculosis
Alternatives
Determination of unusual activity of bacteriocins
Effect on Mycobacterium tuberculosis
Inhibitory growth effect of Bacteriocins on Mycobacterium tuberculosis clinical strain.
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Alternatives