Mycobacterium ----Tuberculosis

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					Mycobacterium
       MYCOBACTERIUM
THIS GENUS IS COMPOSED OF:
   Strictly aerobic, acid-fast rods, does not
   Stain well (gram stain indeterminant),
   DNA has high g+c content, unique cell wall,
   Mycolic acid carbon chain length > c60
   Relatively 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 GROUPS



     1. THE TUBERCULOSIS COMPLEX

     2. SLOW GROWING MYCOBACTERIA
        OTHER THAN TUBERCULOSIS (MOTT)

     3. RAPIDLY GROWING MYCOBACTERIA

     4. MYCOBACTERIUM LEPRAE
                      Acid Fastness 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 COMPLEX

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



         1. M. tuberculosis
         2. M. bovis
Mycobacterium tuberculosis
                        M. tuberculosis
                          General 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.
   COLONIAL MORPHOLOGY OF THE
TUBERCULOSIS COMPLEX MYCOBACTERIA




EUGONIC GROWTH 14 DAYS       DYSGONIC GROWTH 14 DAYS
Mycobacterium tuberculosis     Mycobacterium bovis
           Resistance:
    Not sensitive              Sensitive
     Dry (highly)                Wet
Chemical disinfectants   Heat(62-63℃,15min)
      (more)

 3%HCL, 6%H2SO4,         Alcohol (to nonspore-
  4%NaOH (15min)          forming bacteria)

     Malachite                   UV
   green(1:13000)
Transmission


   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:


Primary infection: children
Secondary infection: age>25
Virulence 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
                   Lipid
Lipid: closely related to virulence
a. Phospholipid
   monocytes proliferate,cause tubercles
b. Wax D
    adjuvent(not only to TB), delayed-type
     hypersensitivity
c. Sulfatide硫酸脑苷脂
    suppress phagosome combine with lysosome
d. Cord factor (trehalose-6,6-dimycolate)
     destroy mitochondria, cause chronic
     granulomatosis, suppress WBC wandering
         Pathogenesis

                    primary infection
1) lung infection
                    secondary infection

2) Out lung infection
     Clinical 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.
           Epidemiology

high epidemic area : babies and children

low epidemic area : elderly people
Mortality

g High

g Moderate

g None or low
Primary 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 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 acellular
    masses of caseous materials.
    (caseous lesion)
粟粒性肺结核
    图注:肺组织内可
    见多个灶性粉染无
    结构结节——结核
    性肉芽肿。中央组
    织彻底坏死,可见
    核缩、核碎现象。
    周围有组织细胞增
    生形成上皮样细胞
    和郎罕巨细胞,淋
    巴细胞散在浸润。
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
        肾结核




结核的坏死破坏到肾盂组织,形成与输尿管道相
 同的腔洞,坏死组织随尿排出体外形成空洞。
图注:1.结核巨细胞(郎罕细胞),胞体巨大,外
形不规则。胞浆灰蓝略带紫色,边缘部有少许空泡
且不整,并围以淋巴细胞形成栏栅状。浆中含有上
皮样肾形核约60个,个别核为圆形,排列紊乱。核
染色质细致,有者隐约有核仁/。其余增色为淋巴细
           胞。
结核性肉芽肿是由于结核杆菌感染引起的具有诊断意义
的病变。中央为干酪样坏死,周围为增生的上皮样细胞,
其内散在Langhans巨细胞,外围聚集的淋巴细胞。
            于上皮样细胞
            及组织细胞,
            淋巴细胞间,
            可见郎罕氏巨
            细胞及残留骨
            组织。




股骨远侧干骺端骨质
破坏与硬化,其中有
散在死骨。病变穿过
骺板累及关节,关节
囊肿胀。
PULMONARY TUBERCULOSIS
                      TUBERCULOSIS
Large caseating tubercle     Miliary tubercles




HUMAN LUNG                   HUMAN LUNG
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)
              Steps in the development of tuberculosis
                         Inhalation of bacteria       Dead
                                                      phagocytes,
                        Bacteria reach lungs,         necrosis
                        enter macrophages                           M. tuberculosis


                             Bacteria reproduce
                             in macrophages


                         Lesion begins to form                      Phagocytes,
                         (caseous necrosis)                         T cells, and
       Activated                                                    B cells
       macrophages                                                  trying to
                                                                    kill bacteria
    Bacteria cease to
    grow; lesion calcifies               Lesion         Bacteria coughed
                                         liquefies        up in sputum
  Immune
suppression
                                         Spread to
                                       blood organs
      Reactivation

                                           Death
          Immunity
High rate of infection, but low morbidity.

Nonspecific immune
AIDS, immunosuppressive agents,
endocrine disease, etc.
        Immunity-cellular Immunity
First time: TB invade→proliferate on the spot →invade
  local lymph node
Macrophage engulf TB
→TH cell
→IL-1 → TH proliferate →bloodstream
Then TH meet TB again
→MCF →macrophages congregate to focus
→MAF →macrophages become more active
→MIF →macrophages stay at the focus
Then if it is successful granulomatosis forms,prevent TB
  diffusing;If it is not successful,macrophage can not kill
  TB, patients deteriorate.
              Immunity
  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.
        Immunity
IV hypersensitivity
Koch phenomenon;
wax D+tuberculin protein;
wax D →macrophage→epithelioid
cell→tubercles→protect TB being
phagocytized
           Immunity
 Humoral immunity
 A lot of Ab comes out, but meaningless
TB active patient: immune complex more
TB stable patient: immune complex less
          Diagnosis
The 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
            Diagnosis
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 test
PPD-C
BCG-PPD
>5mm +
>15mm + +
PPD-C>BCG-PPD infected
Mantoux method
When 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.
       Prevention
BCG vaccination for new infants
 Freeze-drying vaccine
 rRNA vaccine
eg:south India Chingleput’s failure
of BCG
Find and cure patients
          Treatment for Tuberculosis


   Chemotherapy
    treated with a combination of multiple
    drugs for a long period of time: rifampin,
    isoniazid (INH), pyrazinamide, ethambutol,
    and streptomycin.
     Emergent Problems of Tuberculosis

   Emergence of multi-drug resistant M.
    tuberculosis strains.
Mycobacterium avium and
         AIDS
    Mycobacteria and AIDS
• M. avium is much less virulent than M. tuberculosis
   – does not infect healthy people
   – infects AIDS patients

• M. avium infects
   – when CD4 count greatly decreased

• M. tuberculosis infection
   – infects healthy people

   – infects AIDS patients
      * earlier stage of disease
      * more systemic
Clinical features with AIDS

 • systemic disease (versus pulmonary)
    – greater in AIDS

 • lesions often lepromatous
               Antibiotic therapy

• selected primarily for M. tuberculosis
• if M. avium involved other antibiotics included
        Mycobacterium avium-
        intracelluare 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 ulceranns
 does not grow above 33oc
    causes burui ulcer, emerging infectious
  disease
    infection limited to fatty tissue beneath
  dermis
    Mycobacterium marinum
 extrapulmonary ulcerative lesions
   growth of organism restricted to 34oc
   disease called “swimming pool
  granuloma”
   does not respond well to therapy
   Runyon group I
     Mycobacterium kansasii
 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 scrofulaceum
   causes scrofula (cervical lymphadentis)
   drug resistant
   Runyon group II (scotochromogen)
     Mycobacterium fortuitum
           complex
 causes chronic abscesses
   (often wound associated)
   can be confused with M. tuberculosis
   often drug resistant
   rapidly growing (Runyon group IV)
Mycobacterium leprae
 HANSEN’S DISEASE (Leprosy)
     caused by M. 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 forms of Leprosy

   Lepromatous
   Tuberculoid
   Borderline
   indeterminate
ulcers, resorption of bone
worsened from careless use of hands (nerve damage)