MycobacteriumMycobacterium

MYCOBACTERIUMMYCOBACTERIUMMYCOBACTERIUMMYCOBACTERIUM

THIS GENUS IS COMPOSED OF: Strictly aerobic, acid-fast rods, does notStain well (gram stain indeterminant),DNA has high g+c content, unique cell wall,Mycolic acid carbon chain length > c60Relatively slow growth (two groups)

A. RAPID GROWERS (Visible colonies in <5 days)B. SLOW GROWERS (Visible colonies in > 5

days)TYPE SPECIES: Mycobacterium tuberculosis

THE GENUS MYCOBACTERIUM CAN BE DIVIDED INTO FOUR BROAD GROUPSTHE GENUS MYCOBACTERIUM CAN BE DIVIDED INTO FOUR BROAD GROUPS

1. THE TUBERCULOSIS COMPLEX

2. SLOW GROWING MYCOBACTERIA OTHER THAN TUBERCULOSIS (MOTT)

3. RAPIDLY GROWING MYCOBACTERIA

4. MYCOBACTERIUM LEPRAE

Acid Fastness StainAcid Fastness Stain(Ziehl-Neelsen stain)(Ziehl-Neelsen stain)

flood the slide with basic fuchsin (a red dye) in 5% phenol as a mordant.

heat gently for few minutes to melt the wax.

wash with 3% HCl in ethanol. counter-stain with methylene

blue.

Mycobacterium stains red and other bacteria and the background are blue. The mycolic acid and its derivatives are responsible for the acid f

THE TUBERCULOSIS COMPLEXTHE TUBERCULOSIS COMPLEX

(Organisms that resemble M. tuberculosis;Causing a similar type of disease in humans)

1. M. tuberculosis

2. M. bovis

Mycobacterium tuberculosisMycobacterium tuberculosis

MM. . tuberculosistuberculosisGeneral FeaturesGeneral Features

It is a causative agent for human tuberculosis.

It grows very slow with a generation time of 12-15 hours.

On solid media the colonies are raised and rough with a wrinkled surface.

M. tuberculosis cells grow either as discrete rods or as aggregates. Virulent strains tend to grow as an aggregated long arrangement called serpentine cord. Cord factor is a derivative of mycolic acids, trehalose 6'-dimycolate.

ResistanceResistance::

UVMalachite green(1:13000)

Alcohol (to nonspore-forming bacteria)

3%HCL, 6%H2SO4,

4%NaOH (15min)

Heat(62-63℃,15min)Chemical disinfectants (more)

WetDry (highly)

SensitiveNot sensitive

EUGONIC GROWTH 14 DAYS DYSGONIC GROWTH 14 DAYS

Mycobacterium tuberculosis Mycobacterium bovis

COLONIAL MORPHOLOGY OF THE

TUBERCULOSIS COMPLEX MYCOBACTERIA

COLONIAL MORPHOLOGY OF THE COLONIAL MORPHOLOGY OF THE

TUBERCULOSIS COMPLEX MYCOBACTERIATUBERCULOSIS COMPLEX MYCOBACTERIA

TransmissionTransmission

Through respiratory tract, alimentary tract, injured skin 。TB in the lungs or throat can be infectious. This means that the bacteria can be spread to other people. TB in other parts of the body, such as the kidney or spine, is usually not infectious.

Who is at risk:Who is at risk:

Primary infection: children

Secondary infection: age>25

Virulence factorsVirulence factors No spore, no flagellum, no exotoxin,no

endotoxin, no invasive enzyme

Capsule:polysaccharide;CR3;enzyme; protect

Lipid/Lipo arabinomannan

Heat-shock protein/Tuberculin protein: antigenicity, old tuberculin; associate with wax D can cause hypersensitivity and form tubercle

LipidLipid

Lipid: closely related to virulencea. Phospholipid monocytes proliferate,cause tuberclesb. Wax D adjuvent(not only to TB), delayed-type

hypersensitivityc. Sulfatide硫酸脑苷脂 suppress phagosome combine with lysosomed. Cord factor (trehalose-6,6-dimycolate) destroy mitochondria, cause chronic

granulomatosis, suppress WBC wandering

PathogenesisPathogenesis

primary infection

1) lung infection

secondary infection

2) Out lung infection

Clinical syndromesClinical syndromes

a. fatigue, weakness, weight loss and fever

b. pulmonary involvement: chronic cough,spit blood

c. meningitis or urinary tract involvement

d. bloodstream dissemination: miliary tuberculosis with lesions in many organs and a high mortality rate.

Primary TuberculosisPrimary Tuberculosis

The organisms are transmitted among human via aerosol.

TB bacilli lodge in the alveoli or lung alveolar ducts and most of bacilli are phagocytosed by alveolar macrophages.

Macrophages migrate to the hylar lymph node and generate T cell-mediated immune response.

(can be monitored by tuberculin test)

Tuberculin Skin TestTuberculin Skin Test

Tuberculin is a mixture known as purified protein derivatives (PPD) from TB bacilli.

It is a test for delayed type hypersensitivity. Positive reaction, reddening and thickening (> 5mm) at the site of injection after 2-3 days, indicates cellular immunity to tubercle bacilli.

Macrophages containing TB bacilli clump together and begin to form tubercles. (granulomatous response)

With time, the centers of the tubercles become necrotic and form cheesy acellularmasses of caseous materials. (caseous lesion)

Symptoms:Activation of macrophages -> cytokine secretion, IL-1: fever,

TNF: lipid metabolism, weight loss, tissue necrosis. Oxygen radicals: tissue damages

Tissue necrosis -> inflammation -> mucous secretion, destruction of

blood vessels -> frequent cough and bloody sputum

PULMONARY TUBERCULOSISPULMONARY TUBERCULOSISLarge caseating tubercle Miliary tubercles

HUMAN LUNGHUMAN LUNG HUMAN LUNGHUMAN LUNG

TUBERCULOSISTUBERCULOSIS

MYCOBACTERIUM TUBERCULOSIS MYCOBACTERIUM TUBERCULOSIS

Can infect (disseminate) and cause disease

in many different body locations such as:

1. Meninges

2. Brain

3. Bone

4. Kidney

5. Essentially any organ (lung primary target)

Bacteria coughedup in sputum

Inhalation of bacteria

Bacteria reach lungs,enter macrophages

Bacteria reproducein macrophages

Lesion begins to form(caseous necrosis)

Activatedmacrophages

Bacteria cease togrow; lesion calcifies

Immunesuppression

Reactivation

Lesionliquefies

Deadphagocytes,necrosis

M. tuberculosis

Phagocytes,T cells, andB cellstrying tokill bacteria

Death

Spread toblood organs

Steps in the development of tuberculosis

ImmunityImmunity High rate of infection, but low morbidity. Nonspecific immune

AIDS, immunosuppressive agents, endocrine disease, etc.

Immunity-Immunity-cellular Immunitycellular ImmunityFirst time: TB invade→proliferate on the spot →invade

local lymph nodeMacrophage engulf TB →TH cell→IL-1 → TH proliferate →bloodstreamThen TH meet TB again→MCF →macrophages congregate to focus→MAF →macrophages become more active→MIF →macrophages stay at the focusThen if it is successful granulomatosis forms,prevent TB

diffusing;If it is not successful,macrophage can not kill TB, patients deteriorate.

ImmunityImmunity

Cellular immunity

3-6 weeks, T cell VS macrophage

1. CD4+TH : INF-γ→macrophage→epithelioid cell granulomatosis

2. CD8 +TS : granule dependent, dissolve infected macrophage,kill TB

3. CD4- CD8 –t(γδ-T):Fas dependent, dissolve infected macrophage,but not kill TB, cause caseous focus in the center of granulomatosis; Acidity and lack of oxygen also make TB die.

ImmunityImmunity

IV hypersensitivity

Koch phenomenon;

wax D+tuberculin protein;

wax D →macrophage→epithelioid cell→tubercles→protect TB being phagocytized

ImmunityImmunityHumoral immunity

A lot of Ab comes out, but meaningless

TB active patient: immune complex more

TB stable patient: immune complex less

DiagnosisDiagnosisThe steps to diagnose TB infection and

disease include: A medical evaluation that includes

history and risk assessment The tuberculin skin test A chest x-ray A bacteriological examination

DiagnosisDiagnosis

1. Specimen: sputum, pus, CSF, urine, etc.

2. Microscopic examination: Ziehl-Neelsen stain

3. Concentration: 4%NaOH-3%HCL; 6% H2SO4

4. Culture:

solid culture (2-4 weeks 37 ) ;℃ liquid culture (1-2 weeks)

5. Animal inoculation: guinea pig

6. quick Diagnosis: PCR

Skin testSkin test

PPD-C

BCG-PPD

>5mm +

>15mm + +

PPD-C>BCG-PPD infected

Mantoux methodMantoux methodWhen the Mantoux skin test is performed, a

needle is injected into the upper skin layer of the patient's arm. The arm is examined 48 to 72 hours after the tuberculin injection in order to evaluate the reaction on the patient's skin. Any swelling that can be felt around the site of the injection, also known as induration, is measured. The diagnosis of TB infection depends on the size of the measured induration and the patient's individual risk factors.

PreventionPreventionBCG vaccination for new infants

Freeze-drying vaccine

rRNA vaccine

eg:south India Chingleput’s failure of BCG

Find and cure patients

Treatment for TuberculosisTreatment for Tuberculosis

Treated with a combination of multiple drugs for a long period of time: rifampin, isoniazid (INH), pyrazinamide, ethambutol, and streptomycin.

Emergence of multi-drug resistant M. tuberculosis strains.

Mycobacterium aviumMycobacterium avium and and AIDSAIDS

• M. avium M. avium is much less virulent than is much less virulent than M. tuberculosisM. tuberculosis– does not infect healthy peopledoes not infect healthy people– infects AIDS patientsinfects AIDS patients

• M. aviumM. avium infects infects– when CD4 count greatly decreasedwhen CD4 count greatly decreased

• M. tuberculosisM. tuberculosis infection infection– infects healthy peopleinfects healthy people

– infects AIDS patientsinfects AIDS patients* earlier stage of diseaseearlier stage of disease* more systemicmore systemic

Mycobacteria and AIDSMycobacteria and AIDS

• systemic disease (versus pulmonary)systemic disease (versus pulmonary)– greater in AIDS greater in AIDS

• lesions often lepromatouslesions often lepromatous

Clinical features with AIDSClinical features with AIDS

Antibiotic therapyAntibiotic therapy

• selected primarily for selected primarily for M. tuberculosis M. tuberculosis • if if M. aviumM. avium involved other antibiotics included involved other antibiotics included

Mycobacterium avium-Mycobacterium avium-intracelluareintracelluare complex complex

causes tb like disease in birds, opportunistic pathogen in humans. Very prominent cause of disease in aids patients has been decreased following haart. Not easily transmitted. (Runyon group III). Difficult to treat ( drug of choice is rifabutin)

Mycobacterium Mycobacterium fortuitumfortuitumcomplexcomplex

Causes chronic abscesses (often wound associated)

Can be confused with M. tuberculosis

Often drug resistant

rapidly growing (Runyon group IV)

Mycobacterium Mycobacterium kansasiikansasii

Pulmonary and disseminated disease similar to tuberculosis (organisms do not produce niacin)

does not respond well to antimicrobials, (no response to anti-tuberculosis therapy)

Opportunistic pathogen

Runyon group I (photochromogen)

Mycobacterium Mycobacterium marinummarinum

Extrapulmonary ulcerative lesions

Growth of organism restricted to 34oc

Disease called “swimming pool granuloma”

Does not respond well to therapy

Mycobacterium Mycobacterium ulcerannsulceranns

Does not grow above 33oc

Causes burui ulcer, emerging infectious disease

Infection limited to fatty tissue beneathdermis

Mycobacterium lepraeMycobacterium leprae

HANSEN’S DISEASE (Leprosy) HANSEN’S DISEASE (Leprosy) caused by caused by M. lepraeM. leprae

Hansen’s disease is a chronic, slowly progressive granulomatous disease involving ectodermally

derived tissue such as the skin and peripheral nerves. The

disease is usually limited to the cooler parts of the body such as the skin, nose and upper respiratory tract. It rarely affects internal organs such as the brain, liver, spleen, kidneys, and bones.

It has a specific predilection for peripheral nerves.

4 4 forms of forms of Leprosy: Leprosy: LepromatousLepromatous Tuberculoid Tuberculoid Borderline Borderline indeterminate indeterminate