Mircrobiology Lecture -17 Mycobacteria

Mycobacteria Tuberculosis

Tuberculosis

is necrotizing bacterial infection with waid distribution. The lungs are most common by affected (75%) pulmonary tuberculosis. Extra pulmonary tuberculosis includes: meningitides, osteomyelitis, miliary tuberculosis (multisystem infection – associated with night sweats, fever and weight loss), cervical and mesenteric lymhadenoparthy, abdominal

According

to the World Health Organization (WHO), nearly 2 billion people—one third of the world's population—have been exposed to the tuberculosis pathogen., 8 million people become ill with tuberculosis, and 2 million people die from the disease worldwide. In 2004, around 14.6 million people had active TB disease with 9 million new cases. The annual incidence rate varies from 356 per 100,000 in Africa to 41 per 100,000 in the

M.

tuberculosis, then known as the tubercle bacillus, was first described on 24 March 1882 by Robert Koch, who subsequently received the Nobel Prize in physiology or medicine for this discovery in 1905; the bacterium is also known as Koch's bacillus.

 Mycobacterium

can be classified into several major

groups  M. Tuberculosis -95%  M. Bovis -3-5% (M. Bovis infection is typically localized to bone marrow and cervical or mesenteric lymph nodes).  M.africanum,-2-3%;  M. leprae which causes Hansen's disease or leprosy;  Nontuberculous mycobacterium (NTM) are all the other mycobacterium which can cause pulmonary disease resembling tuberculosis, lymphadenitis, skin disease, or disseminated disease.  Clinical infection due to other atypical mycobacterium is rare.

The

genus Mycobacterium consist of non motile, non sporforming, non-capsulated, obligate aerobic. Classification is based on culture characteristics, including nutritional requirement, rate of growth, pigmental and biochemical properties.

Classification

is based on culture characteristics, including nutritional requirement, rate of growth, pigmental and biochemical properties. Mycobacteria cannot be classified as either G+ or G-, and cannot be decolorized by alcohol, but quickly decolorized by 3% hydrochloric acid.

The

genome of the H37Rv strain was published in 1998. Its size is 4 million base pairs, with 3959 genes. The genome contains 250 genes involved in fatty acid metabolism, with 39 of these involved in the polypeptides metabolism generating the waxy coat. Such large numbers of conserved genes shows the evolutionary importance of the waxy coat to pathogen survival.

M.

tuberculosis is an obligate aerobe (weakly Gram-positive mycobacterium, hence ZiehlNeelsen staining, or acid-fast staining, is used). While mycobacterium do not seem to fit the Gram-positive category from an empirical standpoint (i.e., they do not retain the crystal violet stain), they are classified as acid-fast Gram-positive bacteria due to their lack of an outer cell membrane

All

Mycobacterium species share a characteristic cell wall, thicker than in many other bacteria, which is hydrophobic, waxy, and rich in mycolic acids/mycolates. The cell wall consists of the hydrophobic mycolate layer and a peptidoglycan layer held together by a polysaccharide, arabinogalactan.

M.

tuberculosis grow very slowly M. tuberculosis divides every 15-20 hours, which is extremely slow compared to other bacteria, which tend to have division times measured in minutes (E. coli) can divide roughly every 20 minutes).Isolation of the slowgrowing organisms can require 3 to 8 weeks of incubation.

It

is a small bacillus that can withstand weak disinfectants and can survive in a dry state for weeks. Its unusual cell wall, rich in lipids (e.g., mycolic acid), is likely responsible for this resistance and is a key virulence factor They are unusually resistant to killing by phagocytes. They are also higly resistant to drying, but they are killed during pasteurization of milk (heating to 60 C for 30 minutes).

 The

slow growth results from inability to transport nutrients rapidly across the wax layer.  Slow growth causes delay in diagnosis by laboratory  Cultures of clinical material are incubated for up to 8 weeks.The doubling time of tubercle ,acilli is about 18 hours.  On agar, colonies of mycobacterium look like irregular waxy lumps.  The pigmented mycobacterium produce yellow carotinoids.  Colonies of mycobacterium cannot be dispersed 

colonies of mycobacterium

Colonies of mycobacterium

 Cell

wall of M.tuberculosis composed of 80% of water, 20 % of dry substances.  Proteins are presented by 9 tuberculoprotein.  The most important of them is tuberculin. The immune response is aimed against this antigen. Immunization of sensitized organism by tuberculin causes the allergic reaction. Tuberculin  of the host modify them changing their antigen structure. Eextracted and purified preparations of these protein derivatives are used as a skin test reagent to measure exposure

 Polysaccharides

are presented arabinogalactate which ensure immunogenicity of mycobacterium.

L

by the

lipid s are presented by there groups. First group – acetonsoluble lipids – can inter in cell membrane of the host modify them. Second – phosphatides are nonantigenici. Inoculation of phospatides in laboratory animals caused to formation of caverna cavity.

Virulence factor Wax

C and Wax D Wax C – is cord-factor. It consist of micol acid and thregalose. It is adhesive factor to macrophages, antiphagocytic factor at the some time. Wax D forms the additional layer and violate the metabolism of the host cell. That is the reason why the mycobacterium grow slowly.

Wax C form virulrnt strains of tubercle bacillli “serpentine cord” in which acid-fast bacilli are arrenged in parallel chains.

Virulence factor (conclusion) Wax

C – is cord-factor, antiphagocytic factor and factor adhesive to macrophages Wax D - antiphagocytic factor. Tuberculin - diffuse the immune response. Acetonsolable lipids –which modify host cell membrane and diffuse the immune response.

Epidemiology.  Infection

is transmitted by inhalation of Mycobacterium tuberculosis in aerosols and dust.  M. tuberculosis is transmitted from person to person by respiratory aerosol, initial site of infection is the lung.  Air-borne transmission of tuberculosis is efficient because infected people cough up enormous numbers of mycobacterium, projecting them into the environment, where their waxy outer coat allows survive for long periods of time in air and house dust.

 Human

are the natural reservoir of M. tuberculosis. Most transmission occurs by aerosols generated by the coughing.  Initial site of infection is the lung.  M. bovis is found in cow’s milk,(unpasteurized) can cause tuberculosis in human.  Tubercle bacilli do not produce exotoxin or endototoxin.  The severe manifestation of tuberculosis are linked to host reactions to the organisms, damage is caused by uncontrolled, progressive, chronic inflammation and by organism living with macrophages.

M.tuberculosis

infects macrophages and spread of the organism within the body occurs by two mechanism. 1.A tubercle can erode into a bronchus and spread the organism to other part of the lungs, To gastrointestinal tract. 2.It can disseminate via bloodstream to many internal organs.

Macrophages

are the primary cells infected by M. tuberculosis. Early in infection, tuberculosis bacilli replicate essentially unchecked, while later in infection, the Thelper response stimulates macrophages to contain the proliferation of the bacteria.  M. tuberculosis enters macrophages by endocytosis mediated by several macrophage receptors: mannose receptors bind lipoarabinomannan, a glycolipid in the bacterial cell wall.

M.

tuberculosis replicates within the phagosome by blocking fusion of the phagosome and lysosome, mycobacterium block phagolysosome formation.  M. tuberculosis has several mechanisms for blocking phagolysosome formation, including inhibition of Ca2+ signals and blocking recruitment and assembly of the proteins which mediate phagosome-lysosome fusion.

Thus

the earliest stage of primary tuberculosis (<3 weeks) in the no sensitized individual is characterized by proliferation of bacteria in the pulmonary alveolar macrophages and airspaces, with resulting bacteremia and seeding of multiple sites. Despite the bacteremia, most patients at this stage are asymptomatic or have a mild flulike illness.

About

3 weeks after infection, a TH1 response against M. tuberculosis is mounted that activates macrophages to become bactericidal. TH1 cells are stimulated by mycobacterium antigens drained to the lymph node, which are presented with class II major histocompatibility proteins by antigen presenting cells. Differentiation of TH1 cells depends on the presence of IL-12, which is produced by antigen presenting cells that have encountered the mycobacterium.

Mature

TH1 cells, both in lymph nodes and in the lung, produce IFN-γ. IFN-γ is the critical mediator which drives macrophages to become competent to contain the M. tuberculosis infection. IFN-γ stimulates formation of the phagolysosome in infected macrophages.  IFN-γ also stimulates expression of inducible nitric oxide syntheses (iNOS), which produces nitric oxide (NO). NO generates reactive nitrogen intermediates and other free radicals capable of oxidative destruction of several mycobacterium constituents, from cell wall to DNA.

In

addition to stimulating macrophages to kill mycobacterium, the TH1 response orchestrates the formation of granulomas and caseous necrosis. Activated macrophages, stimulated by IFN-γ, produce TNF, which recruits monocytes. These monocytes differentiate into the "epithelioid histiocytes" that characterize the granulomatous response. In many people, this response contains the bacteria and doesn't cause significant tissue destruction or illness. In other people, the infection progresses due to age or immunosuppression, and the ongoing immune response results in tissue destruction due to caseation and cavitation.

Granuloma

Гигантские клетки

The

combination of a single lesion in the lung and caseation in the bronchial lymph nodes is called the Ghon complex. Primary tuberculosis –is the disease of person who are infected for the first time. Primary tuberculosis may take two courses. In people who are otherwise healthy, the lesions become fibrotic or calcified. These lesion usually persist for a life time and can be seen years later in chest x-rays.

Ghon complex.

Caverna

In

immunocompromised person, the organisms may invade the bloodstream and cause disease in almost any organ of the body. This can lead to a potentially fatal generalized infection -disseminated miliary tuberculosis.

Tubercles

are visible in many organs, including the liver, spleen, kidneys, brain and meninges.

Miliary tuberculosis.

The

name “miliary” is derived from the millet. The importance of TNF in this response is underscored by the fact that patients with rheumatoid arthritis who are treated with a TNF antagonist have an increased risk of tuberculosis reactivation.

In

summary, immunity to M. tuberculosis is primarily mediated by TH1 cells(T-lymphocytes), which stimulate macrophages to kill the bacteria. This immune response, while largely effective, comes at the cost of hypersensitivity and the accompanying tissue destruction

Reactivation

of the infection or reexposure to the bacilli in a previously sensitized host results in rapid mobilization of a defensive reaction but also increased tissue necrosis. Just as hypersensitivity and resistance appear in parallel, so, too, the loss of hypersensitivity (indicated by tuberculin negativity in a previously tuberculin-positive individual) may be an ominous sign that resistance to the organism has faded.

Two

substances produced by macrophages, interleukin -1 and tumor necrosis factor are know to contribute to the symptoms of the disease. Interleukin -1 acts as the mediator of the fever experienced by patients with tuberculosis. Tumor necrosis factor or cachectin interferes with lipid metabolism and lead to severe weight loss.

Humoral

immunity does not play a major role in the immune response to tuberculosis. Antibodies appear in the circulation but do not seem to play an effective defensive role, they useful as a diagnostic tool.

Complications of Mycobacterium tuberculosis infection arise from local spread or dissemination Body The

organism may disseminate via the lymphatic's and bloodstream to other parts of the body. This usually occurs at the time of primary infection, and in this way chronic foci are established, which may proceed to necrosis and destruction in, for example, the kidney. Alternatively, spread may be by extension to a lung, into a bronchus, or into the pleural

Clinical fingins  Gastrointestinal

TB – is characterized by abdominal pain and diarrhea accompanied by more generalized symptos of fever and weight loss.  Intestinal obstruction or hemorrhage may occur.  The ileocecal region is the site most often involved.  Tuberculosis of GI can be caused by either M.tuberculosis when it is swallowed after coughed from a lung lesion or by M.bovis when it is ingested in unpasteurized milk products.  In renal tuberculosis –dysuria, hematuria,”flank pain”  “Sterile pyuria”. The urine contains white blood

diagnosis of tuberculosis A

diagnosis of tuberculosis is suggested by the clinical signs and symptoms referred to above, supported by characteristic changes on chest radiography and positive skin test reactivity in the tuberculin (Mantoux) test.  Microscopic examination of a smear of sputum stained by Ziehl-Nielsen's method or by auramine often reveals acid-fast rods.  This result can be obtained within 1 h of receipt of the specimen in the laboratory. This is important because Mycobacterium

Sputum stained by ZiehlNielsen's method

Sputum stained by ZiehlNielsen's method

Sputum stained by auramine

Sputum stained by auramine

diagnosis of tuberculosis Rapid

non-culture tests to detect mycobacteria, e.g. using the polymerase chain reaction (PCR).

Skin test.

Tuberculin

skin test is most widely used to diagnose tuberculosis. It only detects delayed hypersensitivity, and does not indicate the presence of active disease. The tuberculin skin test is perfomed by injecting a small amount of PPD, a mixture of proteins from tubercle bacilli. A positive test is indicated by reddening and thickening (indurations) of skin 48-72 hours after injection. This is due to infiltration of the area by mononuclear phagocytes and T-cells.

Skin test.

This

delayed-type hypersensitiiivity reaction reflects the local evens that take place in the infected tissue.

Specific antituberculous drugs Mycobacterium

are innately resistant to most antibacterial agents, and specific antituberculous drugs have to be used; these are reviewed in. combination therapy usually three drugs such as isoniazid, rifampicin, ethambutol to prevent emergence of resistance prolonged therapy - minimum 6 months period which is necessary to eradicate these slow-growing intracellular organisms.

Tuberculosis is prevented by improved social conditions, immunization and chemoprophylaxis Immunization with a live attenuated

BCG (bacilli Calmette-Guérin) vaccine, has been used effectively in situations where tuberculosis is prevalent. Immunization, which confers positive skin test reactivity, does not prevent infection, but it allows the body to react quickly to limit proliferation of the organisms. In areas where there is a low prevalence of disease, immunization has been largely replaced by chemoprophylaxis.

Prophylaxis

with isoniazid for 1 year is recommended for people who have had close contact with a case of tuberculosis. It is also advocated for individuals who show recent conversion to skin test positivity, when it is essentially early treatment of subclinical infection rather than prophylaxis.

“Atypical” mycobacteria 

   



Atypical” mycobacteria are classified into four groups according to their rate of growth and whether they produce pigment under certain condition. Group I organism (Photochromogens) produce a yellow-orange-pigmented colony only when exposed to light. M.kansasii causes lung disease clinically resembling tuberculosis. M.marinum causes “swimming pool granuloma”. These granuloma, ulcerating lesions occur in the skin at the site of abrasion incurred at swimming pools and aquriums. The natural habitat of the organism is both fresh and salt water. Treatment with tetracycline.

“swimming pool granuloma”.

Atypical mycobacteria  Group

II organism(Scotochromogenes) produce the pigment chiefly in dark.  M.scrofulaceum causes scrofula, a granulomatous cervical adenitis in children. The organism enters through the oropharyx and infects the draing lymph nodes.  Its natural habitat is environmental water souces, but it has also been isolated as a saprophyte from the human respiratory tract.

Atypical mycobacteria  Group

III organism(Nonchromogenes)  M.avium-intracellulare complex (MAI,MAC).  Is composed of two species, M.avium and M.intracellulare.  They cause pulmonare disease clinically indistinuishable from tuberculosis, primarily in immunocompromised patients.  The organisns are widespread in the environment, including water and soil.  They are highly resistant to antituberculosis drugs.

Atypical mycobacteria  Group

IV organism (Rapidly Growing Mycobacteria).  M. fortuitum, saprophytes, found in soil and water, rarely cause human disease.  Infection occure in two population:  1.Immunocompromised patients  2.individuals with with indwellig catheters.  They are often resistant to antituberculosis therapy.