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

Zone of inhibition of growth sufficiently large

ATB disc

Growth of tested bacteria

Insufficient zone of inhibition

Without zone of inhibition – resistence to tested ATB

ATB susceptibilityStaphylococcus 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 10-15´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.