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
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
• 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
Microscopy
• Broth cuture of Staphylococcus aureus fixed and stained by Gram:
G+ cocci in clusters
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
Demonstration
G+cocci: 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
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
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
Comparison od atb susceptibility
• Streptococcus pyogenes – HSA /
Staphyolococcus aureus
PNC, TET
• Staphylococcus aureus / Staphylococcus
epidermidis, Staphylococcus haemolyticus
OXA, CEF,
Detection of plasmacoagulase
summer term
• Free
• Bound
• Slide
• Tube method
• Staphylococcus epidermidis
• Staphylococcus aureus
Free coagulase
• Tube method – colony of tested strain is emulsified in
0,5 ml of plasma. Incubated for 6 h.at 37*C 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.
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 1015´sec. Odčítava sa o 10-15 sec.
• White precipitate, agglutination = posit.
• Negative result must be confirmed by tube test
• Fig.
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.