Mycobacterium 2

Mycobacterium sp.
contains lipids in cell wall
nonsporulating aerobe rod
occasionaly producing filaments
cultivation conditions - fastidious
not Gram staining
slowly growing
41species : 27 isolated from men
95% of them caused by
M.tbc, M. avium-intracelulare, M.kansasii, M. foruitum, M. chelonae
M. leprae.
Classification of mycobacteria
Slowly growing
-Mycobacterium tuberculosis complex
Mycobacterium tuberculosis ,
Mycobacterium leprae,
M.bovis, M.ulcerans
Atypical mycobacteriae – classification according Runyon
M.kansasi, M.marinum
M.avium, M.intracelulare
slowly growing non pigmented
M.fortuitum, M.chelonae
quickly growing non pigmented
Mycobacterium – genus
Cell wall
rich for lipids = hydrophobic surface
= resistent for desinfection
= not accepting colour, but if stained
(special procedure – warming sc.Ziehl Neelsen) they
resist to decolorisation by strong acid
= acid resistent, acid fast
Mycobacterium tbc – Ziehl Neelsen sstaining
Growing conditions and colony morphology
complex structure of cell wall and cultivation requirements
Speed of growth and pigmentation – classification sc. Runyon:
Slowly growing, non pigmented – M. tbc......
I. group – fotochromatic – production of intense yellow pigment karotenoid on light
II. group – scotochromatic – production of pigment in dark and light
III. group – slowly growing not pigmented
IV. group – rapidly growing not pigmented
Group of slowly growing and IIIrd group acc. Runyon
- generation time 12 –24 hrs – visible colonies appeat after 3-8 weeks
Group I, II and IV - rapidly growing – first coloniesafter 3 days and
M. leprae – not cultivatable
Cell wall – complex
Peptidoglyc (rigidity of the structure) with phosphodiesterically bound
D - arabinose a D - galactose to
mycolic acid (most important lipids in cell wall).
with free lipids (waxes, mycosides – cord factor) – responsible for
paralel arrangement of bacili in virulent strains
polypeptidmi (PPD) na povrchu.
Cell wall – most outer layer
polypeptides (PPD) on the surface (purified protein derivate)
– antigenic properties,
-- stimulation of cell immunity,
-- extracted and partially purified (PPD) used for skin test
Tuberculin reaction
-- test for specific (anti TBC) cell immunity detection
– hypersensitivity of the IVth type – delayed cell hypersensitivity
Patogenesis of infection
Destruction of tissue and fibrosis – produced by host immunity on
infection by M.tbc
Intracelular bacterium – escape innate immune mechanism
Tuberculosis – classical mycobacterial disease starts after inhalation of
* engulfment by alveolar macrophages
* multiplication intracelularly - escape immunity mechanism and
destoy phagocytes that engulfed them.
* repetition of this process (phagocytosis of bacilli, replication of M.tbc,
lysis of cells = multilobular giant cells of macrophages„Langhans cells“)
* inficated macrophages spread to local lymphatic nodes, blood net,
bone marrow, spleen, kidney, bones, CNS...)
2-4 weeks after infection – specific immunity reaction
Cytokns production by CD4 T lymphocytes aktivise macrophages
and they are then able to kill bacterie localise inside:
low antigenic burden – small tissue devastation
high antigenic burden (masive infection – big tissue devastation-necrosis
Effectivity of bacteria elimination depends on size and localisarion of
localised activated macrophages
(granuloms < 3 mms): macrophages penetrate inside it and kill bakcteria,
eliminate spreading
larger necrosis, caseous granulomas – encircled by fibrin, bacteriae
survive inside, can reactivate after years
Immunity mechanism:
* cytokins produced by T lymfocytes,
* activcation of complement cascade,
* ischemia of tissue ,
* hydrolytic enzymes produced by macrophages
Cell immunity
Reactivation in cell immunity decrease – lack of T lymphocytes:
-immunosupresive therapy – rheumatoid artritis, SM, Crohn disease –
biological therapy to depress cell immunity requires dignosis of latent
Clinical sy and diseases
M.tbc can infect any organ
* lung
* extrapulmonal tbc
* Tbc in HIV infected
* lung (in immunocompromised)
* asymptomatic colonisation
* disseminated infection in HIV positive
M. leprae
* Lepra lepromatosa,
* Lepra tuberculoid
Other mycobacteriosis
* Lung disease (M.kansasií)
* Skin disease (M. marinum,M.ulcerans)
Clinical sy
Lung tbc after inhalation of contaminated aerosol
Inicial lung infection – middle lobe or lower lobe, multiplication 3-6
weeks, untill cell immunity is stimulised
5% infected – acute disease before 2 years
5-10% later in life
Non specific signs, few sputum, rtg signs of lung infections, positive
skin test, laboratory verification microscopically and by cultivation.
Active disease with pneumonia, production of abscesses and cavities
with sever involvement of upper lobes.
Extrapulmonal tbc – hematogenous spread during inicial phases
(nodes, pleura, UGT) – not necessary the lung symptoms
Tbc in HIV positive – decrease of CD4 lymphocytes, present before
other opportunistic infections
Patogenesis and immunity in Mycobacterium leprae
Lepra, Hansen´s disease:
Lepra - lepromatous – strong antibody reaction, defect of cell
immunity to M.leprae as antigen.Many bacteria in skin
macrophages (Schwann cells), very infectious
Lepra - tuberculoid – strong hypersensitivity of delayed type,
feeble humoral immunity, many lymphocytes and granulomas,
few bacteria in tissue (activated macrophages fagocyte and
eliminate bacteriae
Symptoms are caused by immunity reactions
Other mycobacteria:
M.kansasí,M avium-intracellulare
granulomatous inflamation – localised lung infection,
clinicall the same as tbc, or disseminated infection
M.marinum, M.ulcerans: skin infection – prefer lower
M.fortuitum, M.chelonae: very rarely disseminated
Laboratory diagnosis
Microscopy rapid dg, Ziehl Neelsen,
fluorescence, preparation of sample
Cultivation morning sputum 3 days
consequently or a lot of other liquidv– urine
* slow growing
* overgrowing contaminating flora
* decontamination, NaOH,
* egg media(Šula,Lowenstein) and solid
agar media
* rapid dg - detection of metabolism of
palmit acid and CO2 -
Genetic probes detection of aminoacids
specific for M.tbc, shortens dg to 6 hrs,
directly from biol.material
Serology not sensitive nor specific
on common ATB
longlasting therapy (18 –24 mnths lepra., 6-9 mnths other)
= rapid evolution of resistence
= use of combination of antimycobacterial protected ATB
INH, rifampicin, pyrazinamid, ethanbutol, streptomycin + cycloserin
(atypical), chinolons (contaminating infection)
1990 common rezistence in USA in HIV positive
Rapidly growing mycobacteriae
– very resistent on common ATT
- sensitive on aminoglykosides, cephalosporins, TTC, chinolons
M.leprae rapid development of resistence – combination
Antituberculotics and therapy
• Streptomycin (kills actively multiplying M.tbc)
• Izoniazid, Rifampicin (acting on strains in caseouse
• Ethanbutol Etionamid Kanamycin Cycloserin
Pyrazinamid (active intra cellularly)
Pacients woth lung tbc - 3 populations of M.tbc
- localised extracellularly in cavities,
intracellularly in macrophages,
in caseouse necrosis
• Combination of 2-3 ATTs.
Detection of ATB susceptibility
• ATTs are dissolved in cultivation medium, and on the
surface, the tested strain is innoculated
• Method of proportionnal sensitivity – dilution method.
Innoculum is diluted so that cultivation there will be 100300 CFU – colony forming unit – on the medium
• If the number of resistent colonies (growing on the medium
with ATT) is higher then 1% (1-3 colonies) M. tbc – therapy
will be not efficient
• Spontaneous appearance of resistent mycobacteria
without exposition to ANTITUBERCULOTICS
• Frequency of resistent cells is 1:105 in INH and 1:106 in
STM – if the combination is used, incidence of resistent
strains will be 1011.
• Overall population of bacteriae in patients with open
cavity is 10 7 - 109, so there can be as much as 102 - 104
of resistent strains.
• 1 ATT means owergrowing of resistent population of
strains, that will replace sensitive killed bacterial
Combination of 2 and more ATT:
INH+RIF - 9 mnths,
+ STR+ Ethanbutol 2-8 weeks
from 2nd mnth 2x weekly,
INH + Ethanbutol 18-24 months,
Therapy principals
• ATT are active only on multiplying mycobacteria,
• activity of the growth depends on the availability of oxygen
and pH (optimal is neutral or alcaline)
• Max. growth is in open cavities, min. is in caseouse
• i.c. – in fagosomes - pH is 5,5, acid – slow growth and less
bacteria, less resistent strains – ATT supporting acid
environment are used - Pyrazinamide. (STM is not able to
work in fagosomes and loss activity in acid pH)
• in closed caseouse necrosis there is just a few oxygen, slow
metabolism, few living cells, neutral pH and feeble grow
STM – extracelular mycobacteria in tissue
• INH – kills slowly growing cells
• Rifampicin – effective on mycobacteriae in caseouse
necrosis and macrophages
• Pyrazinamide – only in acide pH and macrophages
Ethanbutol – only in combination, able to penetrate in
mycobactera situoated ic and ec
• Capreomycin, Kanamycin – baktericidal for e.c
mycobacteria in cavities
• Ethionamid, cycloserin – bakteriostatic for i.c and e.c
Epidemiology – process of the spread of infection
Man is the only natural host and source
Transmission by longlasting narrow contact with ill or infected
Particles of aerosol with 1-3 tbc bacili can get as far as to alveoli
5-200 bacteriae may cause infections.
of the number of cases, AIDS pacients, alcoholics, narkoman
Resistence on antituberculotics ATTs
Other mycobacteriae
M. bovis – infection of annimals, source of infection of men from milk
M.avium-intraellulare complex – present in water and soil and annimal
Transmission by infected food to immunocompromised patients with
M.leprae 12 milions, 62% in Asia, 34% in Africa, 10% in USA,
transmission by longlasting, premanent contact with infected, by
inhalation, skin contact with nose secresion, exudation from wound
INH 1 year after the contact with M.tbc and positive skin test
atenuated living strain of M. bovis - bacillus Calmette Guerin–BCG strain 5th day after birth,
side effect – BCG-itida
- positive tuberculine skin test ! interpretation!
• Vaccination against tbc
• BCG - Bacillus Calmette - Guérin – vaccination
by life attenuated strains Mycobacterium bovis
– cross reacting Mycobacterium tbc.
• Testovating of cell immunity
- in vivo skin tuberclin test – Mantoux
- in vitro
Tuberculin reaction
i.d 0,1 mcg = 5TU - tuberculin unit
aplication of PPD purified protein derivate of cell wall
after 48 – 72 hrs
<15mms negat – without reaction, anergia ( HIV infected too),
15mms - OK., immunity
more than 15 mms – strong reactions – susp.expositions
IGRA – interferon gama release assay
Quantiferon – in vitro test of specific cell
mediated immunity
• method to detect latent infection
• in vitro detection of specific CMI in blood
• Person exposed in past to antigen has specific T
lymfocytes and memory cells
• after exposition of antigen to whole blood, the
rapid and repeated stimulation of antigen specific
memory cells followed by release of cytokins
follows (interferon gama)
• Detection of released IFN-gama (by ELISA) is a
specific proove of cell mediated reaction to
specific antigen (stimulationg)
Postup pri inkubácii a zbere plazmy
Test is based on exposition of blood in 3 tubes:
- antigen of M.tbc, - v 1 skúmavke
- non specific mitogen – positive control
- negatíve control – without antigen
Whole blood is sampled to 3 tubes – the blood should adhere to
tube wall., after thorought agitation for 5 sec.
Incubation for 16 – 24 hrs at 37 dg.C (untill the memory cells are
sensitised by M.tbc – exposition to specific M.tbc antigen on the
surface of the tube stimulateds secression of INTERFERON
Blood si centrifugated
Plasma is sampled for detection of interferon gama
To detect IF-gama secreted after in vitro activation (antigen on
the surface of the vial + memory cells in whole blood of the
patient) of memory cells of M.tbc and detected by ELISA test
comparison of IFG
in the tube with
-TB ag : negat.contr.
Negat control –
exclude nonspecific
secrection( ex. after
Posit. control –
shows capability to
react (negat. in
or after therapy
Mantoux: in vivo
reaction of memory cells
after BCG and M.tbc.
aplication of PPD i.d.
memory cells after BCG
or M.tbc will go to the
place of innoculation
inflamation and
induration in the place
Quantiferon: in vitro
memory cells after M.tbc
in the tube contact of
memory cell with +
M.tbc = release of gama
IF if memory cells
present. Memory cells
after infection

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