Practicals WT4 Mycobacterium, Corynebacterium

1
Practicals WT4 Mycobacterium, Corynebacterium

• Laboratory diagonosis of mycobacteria ATB
  susceptibility tests
• Laboratory diagnosis of. corynebacterium ATB
  susceptibility test
• Lysogenic conversion
• Antituberculotics
• Microscopy - M.tbc - Ziehl Neelsen, C, diphtheria
  - Gram, Albert, C. pseudodiphthericum - Gram
• Cultivation - M. tbc - Lowenstein and broth
  medium, C. diphtheriae a C. pseudodiphthericum -
  Blood agar and tellurit medium
• Biochemical characteristics of C.diphtheriae a C.
  pseudodiphthericum
• Toxigenicity of C. diphtheria - ELEKs method and
  animal model

2
Laboratory diagnosis of mycobacterium

• Sampling - in pulmonar tbc - morning sputum -3
  consecutive days, extrapulmonar - appropriate
  sample - big quantity of liquid - urine,
  menstrual blood
• Microscopy - Ziehl Neelsen, fluorescence -
  increased sensitivity, preliminary dg
• Cultivation -sample proceeding, decontamination
  of nonmycobacterial flora, condensation of the
  sample - NaOH, centrifugation. Eggs media - ula,
  Lowenstein / solide and broth / - pigment, shape
  - in broth - membrane, sediment - Long generation
  time - quick methods - based on detection of
  metabolits of palmit acid and CO2
• Genetic probes - detection of aminoacids
  sequences

3
Staining

• Ziehl Neelsen - cover heat fixed and dried smear
  with a piece of filtrate papaer. Apply 5-7 drops
  of carbolfuchsine. Heat from beneath till
  vaporisation. Discard paper with forceps, rinse
  and dry. Decolorise with acid alcohol
  (HClethanol) untill the color is stining the
  water. Conterstain with methylen blue. Rinse,
  dry. M. tbc - red, surroundings - light blue.
• Albert - methylen blue or toluidin blue

4
Lab. dg. of corynebacterium

• Diagnosis of the disease is based on clinical
  picture and epidemiological history. Lab. dg. is
  long and complicated Appropriate sampling
• Microscopy - smear with Gram staining and
  Alberts staining does not distinguish between C.
  diphtheriae and other Corynebacteria.
• Cultivation Blood agar - 3 typs od colonies -
  mitis, gravis, intermedius, Lofflers medium,
  tellurit medium brown colonies with halo
• Biochemical identification- differentiation from
  other corynebacteria present in throat
• toxigenicity of the strain - ELEK,
• ATB susceptibility

5
Antituberculotics and therapy

• Streptomycin(kill actively multiplying M.tbc)
  Izoniazid,Rifampicin (effective on M.tbc in
  caseous necrosis) Ethanbutol Etionamid Kanamycin
  Cycloserin
  Pyrazinamid ( active i.c.)
• In pacient with pulmonar tbc - 3 populations of
  M.tbc - localised
  extracelullarly in cavitas,
  -
  intracelullarly in macrophages,
  - inside caseouse
  necrosis
• Combination of 2-3 ATT. Resistence fighting
  -overgrowinf of resistent mutants in culture of
  M.tbc - for INH 1105 for STM 1106 in
  infectious place there is 107- 10 9 of bacteria

6
Lysogenic conversion

• Strain of C. diphtheriae gains his toxigenicity
  by lysogenic conversion - thanks to bacterophage.
  Bacterial virus - bactrophage is able to
  transfere genetic information about the
  production of toxin - tox gen - fom one cell (
  toxigenic) to the other (non toxigenic) C.
  diphtheriae cell.

7
Cultivation of Corynebacteria

• C. diphtheriae - blood agar
• C. pseudodiphthericum - blood agar, inverse CAMP
• C. diphtheriae on tellurit medium - brown
  colonies with brouwn ring - halo

8
Microscopy

• Ziehl Neelsen staining - acid-fast M. tbc - not
  willin to accept stain. If colored - very
  difficult to decolorise
• Gram staining of C. diphtheria - Grods
• Alberts staining - metachromatic staininig of
  granules - resulting color is different from
  color used for staining - C. diphtheriae
• Comparison of Gram smear of C. diphtheriae a C.
  pseudodiphthericum

9
Cultivation of M. tbc

• Egg medium - Lowenstein - colony morphology, no
  pigment
• Broth medium - growing in membrane
• cultivation 3-6 weaks - reading every weak

10
Biochemical characteristics

• Fermentation differentiation of C. diphtheriae
  from other corynebacteria isolated from throat
  (C.ulcerans, C. pseudotuberculosis, C. xerosis,
  C. pseudodiphthericum
• C. diphtheriae
• granul catalase gelatine, urea, lactose,
  maltose, glc
• - -
  -
• -
  - - -
• C. pseudodiphthericum

11
Detection of toxigenicity of C. diphtheriae

• ELEKs test of toxigenicity - imunodiffusion of
  suspension of tested strain and antidiphtheric
  serum in agar - zone of precipitation
• Annimal mode application of
  diphtheria toxin
  i.d. ..necrosis
• antidiphtheric serum (i.p. or i.d.) toxin no
  necrosis

12
ATB susceptibility

• Susceptibility test for antituberculotics - ATT
  are dissolved in medium
• Method of proportional susceptibility testing -
  dilution method. Dilution of inoculum so that
  there is from 100-300 CFU - colony forming units
  on a plate
• If there is more than 1 of M. tbc cells
  resistent to ATT the therapy will not be
  cliniccnally suscessful
• Susceptibility of Corynebacterium - PNC, ERY -
  for elimination of bacteria or carriage state -
  therapy of diphtheria - primarily antitoxic.
  Preventive measures.
• Nondiphtheriae corynebactria - Vancomycin,
  (Cefalosporins of the 1st generation)

13

• Spontaneous appearance of resistent mycobacteria
  - without exposition to ATT
• Frequency of resistent cells in the culture is
  1105 for INH and 1106 for STM - if both are
  given in combination the incidence of resistent
  strains will be 11011.
• The overall population of bacilli in patient with
  open cavity is 10 7 - 109 , so there is 102 - 104
  resistent strains.
• 1 ATT means overgrowing of resistent strains that
  will replace susceptible killed bacteria.
• Combination of 2 and more ATT INHRIF - 9
  months, STR Ethanbutol for 2-8 weaks, from
  2nd months only 2 times weakly, INH Ethanbutol
  18-24 months,

14

• Tests for ATT susceptibility - correlation
  between in vitro and therapy success.
• If more than 1 of M. tbc in vitro are resistent
  - therapy will not be successful
• Tests determine ratio between susceptible and
  resistent strains
• Agar dilution method - depend on innoculum - if
  too thick - there is too much of resistent
  strains - false resistent, if less concentrated -
  false positive - possible resistent strains not
  identified.
• Optimal dilution 100-300 CFU on plate
• Inconvenience - aga can inhibit activity of ATT
  as well as of mycobacteria, long incubation
  decrease activity of ATT

15
Principles of therapy

• Therapy ATT are active only on growing
  mycobacteria, growth depends on oxygen
  availability and pH (optimu neutral or alkaline)
  - Max in open cavity, min in caseouse necrose.
• In i.c. - in phagosomes - pH is 5,5, acid - slow
  growth, less of bacilli, less resistent strains -
  ATT supporting acid environment - Pyrazinamide.
  (STM not able to enter phagosomes and looses
  activity in acid pH)
• in chronic closed caseouse necrose - slow blood
  supply and oxygen, slow metabolism, less living
  cells, pH neutral but slow multiplication

16
ATT

• Bactericidal STM - mycobacteria extracelullarly
  in cavities
• INH - kills slowly and quickly growing cells
• Rifampicin - effective on mycob. Closed in
  caseouse necrosis and macrophages
• Pyrazinamide - only in acid pH and in macrophages
  
• Bacteristatic Ethanbutol - only in combination,
  able to penetrate in mycobacteria situated i.c.
  and e.c.
• Capreomycin, Kanamycin - bactericidal, for e.c
  mycobacteria in cavities
• Ethionamid, cycloserin - bacteriostatic for i.c
  and e.c