Lesson 3 WT Staphylococcal infections

1
Lesson 3 WT
Staphylococcal
infections

• Diagnosis of staphylococcal infections
• Diagnostical model abscess - pus,
  enterotoxicosis - food, osteomyelitis - punctate,
  secretions,
• Microscopy, cultivation, biochemical tests,
  detection of enzym,ATB susceptibility tests

2
Diagnosis of staphylococcal infections

• Microscopy from the base of abscess., in
  aspirations and punctates usually few bacteria
  and more PMNL, blood for hemocultivation as few
  as 1 cell/1 ml, in rest of food in
  enterotoxicosis not sufficient for prooving
• Cultivation - blood agar, salt mannit, pigment,
  hemolysis
• Identification biochemical
• Detection of plasmacoagulase, toxins, enzymes,
  phagotypes, analysis of nucleoacids
• Serology seldome, antibodies against teichooic
  acid - imunodiffusion detection of prolonged
  infection - bacteremia, endocarditis

3

• Abscess - pus - sterile collection
• enterotoxicosis rest of food detection of
  toxin, presence of staphylococcus is not the
  proof
• osteomyelitis - punktate, discharge - sterile
  collection, blood for hemocultivation

4
Microscopy

• Broth cuture of Staphylococcus aureus - fixed and
  stained by Gram
  G cocci in clusters

5
Cultivation of Staphylococci

• Blood agar Staphylococcus aureus - grey to
  yellow pigmented colonies, concave, buttered,
  beta hemolysis
• Stafylococcus epidermidis - white colonies,
  without hemolysis
• Growth on media with NaCl a indicator salt
  manit - only St.aureus changing color of
  indicator from red to yellow

6
Demonstration Gcocci Staphylococcus aureus a
Staphylococcus epidermidis on the
selective-diagnostic medium Salt mannit
selectively NaCl
allowed growing of staphylococci that tolerate
it.while others do not., mannitol is the
diagnostic substrate utilised by St. aureus which
metabolised it, formed acid that makes the
medium becomming acid and change the pH and
indicator color. St. aureus changes the original
red color to yellow, St. epidermidis is growing
on the mediu , tolerates salt without changing
the pH and indicator color - not utilising manitl
7
ATB susceptibility testing

• Disc diffusion method
• 6-8 ATB discs in one plate
• Zone of inhibitionof the growth in mms
  comparison with standards

Without zone of inhibition resistence to tested
ATB
Zone of inhibition of growth sufficiently large
ATB disc
Growth of tested bacteria
Insufficient zone of inhibition
8
ATB susceptibility

• Staphylococcus aureus - PNC - penicilinase,
  semisyntetic PNC oxacilin, methicilin -
  resistence MRSA hospital strains and CoMRSA
• - alteration on the level of target structure -
  pencilin binding protein PBP
• chromosomal type typ connected with
  resistence to other atb - clindamycin,
  erytromycin, aminoglycosides
• Good susecptibility to vancomycine
  transmissible resistance form Enterococcus

9
Comparison od atb susceptibility

• Streptococcus pyogenes HSA / Staphyolococcus
  aureus
• PNC, TET
• Staphylococcus aureus / Staphylococcus
  epidermidis, Staphylococcus haemolyticus
• OXA, CEF,

10
Detection of plasmacoagulase summer term

• Free
• Bound
• Slide
• Tube method
• Staphylococcus epidermidis
• Staphylococcus aureus

11
Free coagulase

• Tube method colony of tested strain is
  emulsified in 0,5 ml of plasma. Incubated for 6
  h.at 37C then at room temperature for 24 hrs.
• Reading after 1 hour, 2 hrs and 24 hrs.
• method watching of coagulum. Formation of
  coagulum pozit.,
• Because of possible presence of fibrinogen in
  plasma this can dissolve the coagulum. That is
  why we read it at 1,2 and 24 hrs. Fig.

12
Bound coagulase

• Slide method
• In 2 drops of steril water or saline solution
  there is the suspension prepared from tested
  strain. 1 drop of plasma is added. Reading after
  10-15sec. Odcítava sa o 10-15 sec.
• White precipitate, agglutination posit.
• Negative result must be confirmed by tube test
• Fig.

13
Detection of catalase and oxidase
activity Catalase - enzyme, hydrolysing H2O2
toxic for the cell and formation of molecular
oxygen. Moraxella catarrhalis cat.negat. H2O2
hydrolysis, bubbles - Staphylococcus sp.