Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download staphylococcus species
Document related concepts
Transcript
BY2012 Microbiology Staphylococcus species Staphylococcus spp. Clusters of Gram-positive cocci Staphylococcus spp. Grape-like Clusters of Gram-positive cocci Blood Agar Plates Staphylococcus aureus Staphylococcus epidermidis Blood Agar Plates Staphylococcus aureus colonies may also be white or cream-coloured and may be haemolytic (depends on species of blood used in blood agar plate Blood Agar Plates Staphylococcus aureus colonies may also be white or cream-coloured and may be haemolytic (may depend on species of blood used in blood agar plate Blood Agar Plates Staphylococcus epidermidis Blood Agar Plates Staphylococcus saprophyticus Staphylococcus capitis Blood Agar Plates Staphylococcus cohnii Staphylococcus xylosus Staphylococcus aureus S. aureus growing on a nutrient agar plate Isolation of Staphylococcus spp. from Anterior Nares Mannitol Salt Agar Plate Selective Agar Medium • Contains 7.5% NaCl [1.28 M, approx 8.5 × isotonic (physiological) saline] – inhibits the growth of most bacteria other than staphylococci • Contains Mannitol, the sugar alcohol of the hexose sugar mannose • Contains Phenol Red as a indicator for acid production from mannitol (change from pink to yellow) Mannitol Salt Agar Plate Selective Agar Medium Staphylococcus aureus Staphylococcus epidermidis Identification of Staphylococcus species • While the source of a staphylococcal isolate can often indicate to an experienced microbiologist its likely identity, staphylococci are not distinguishable by the appearance of colonies on blood agar plates and in Gram-stained preparations • A combination of physiological and biochemical tests have to be performed to reveal the species identity Differentiation of Staphylococci • Coagulase Test • DNase production • Clumping factor test • Sugar fermentation tests Coagulase and Fibrin Clotting Damaged Blood Vessels Exposure of Collagen Fibres Release of Thromboplastin Platelet Activation & Aggregation Prothrombin Inactive Enzyme Coagulase Thrombin Serine protease Staphylothrombin Fibrinogen Fibrin Monomer Soft Fibrin Clot Cross-linked Fibrin Clot Coagulase and Fibrin Clotting The coagulase test uses blood plasma. Blood is collected into an anticoagulant such as citrate or heparin and the red and white blood cells are removed by centrifugation. The clear supernatant (overlying) fluid is blood plasma which contains the fibrinogen needed for the coagulase test Coagulase and Fibrin Clotting When blood is collected in the absence of an anti-coagulant, thromboplastin released from blood platelets activates prothrombin to thrombin which hydrolyses fibrinogen to fibrin giving rise to a blood clot. This removes fibrinogen from the plasma fraction of whole blood. Thus serum can not be used for a coagulase test. Tube Coagulase Test Soft fibrin clot Tube Coagulase Test Cross-linked fibrin clot Fibrinogen ClfB Cytoplasm Collagen Cna Y ClfA Protein A IgG Y Clf = Clumping Factor Clumping Factor Surface Proteins of Staphylococcus aureus Cytoplasmic Membrane Cell Wall FnBPA Fibronectin Single protein molecules anchored to cell wall by flexible “stalk” Clf Clf Surface Proteins are Covalently Linked to Peptidoglycan in Gram-Positive Cocci NAG-NAM L-P-X-T +++ Precursor anchored to membrane G-G-G-G-G- Pentaglycine bridge that normally links 2 peptidoglycan chains NAM, N-acetyl muramic acid NAG, N-acetyl glucosamine GGGGG glycine bridge Schematic Structure of Clumping Factor Fg = Fibrinogen MSCRAMM = Microbial Surface Components Recognising Adhesive Matrix Molecules Clumping Factor S. aureus S. aureus Cross-linking of S. aureus cells by fibrinogen interacting with ClfA and ClfB Fibrinogen Clf B Clf A Clumping Factor Test S. aureus S. epidermidis Fibrinogen causes clumping (aggregation) of S. aureus by crosslinking the bacteria through its interactions with the fibrinogenbinding proteins ClfA and ClfB on its the cell wall Deoxyribonuclease Test High Molecular Weight DNA DNase Mononucleotides Oligonucleotides Precipitates with 1M HCl Do not precipitate with 1M HCl Opacity Clearing Deoxyribonuclease Test – DNA Agar Staphylococcus aureus Staphylococcus epidermidis Zone of clearing (arrowed) No zone of clearing Sugar Fermentation Tests Lactate Production by Staphylococci Other sugars Identification of Staphylococcus Isolates to Species Level by Fermentation Tests Sugar Trehalose Type Disaccharide of glucose (1-O-α-linked) Mannitol Sugar alcohol of mannose Maltose Disaccharide of glucose (4-O-α-linked) Sucrose Disaccharide of glucose and fructose (1-O-α-linked) Xylose Pentose Arabinose Pentose Sugar Fermentation Tests Trehalose Mannitol Maltose Sucrose Xylose Arabinose Staphylococcus aureus Trehalose Mannitol Maltose Sucrose Xylose Staphylococcus epidermidis Arabinose Sugar Fermentation Tests Trehalose Mannitol Maltose Sucrose Xylose Arabinose Staphylococcus saprophyticus Trehalose Mannitol Maltose Sucrose Xylose Arabinose Staphylococcus schleiferi subsp. schleiferi Bioassay of Penicillin with Sensitive Strain of S. aureus The diameters of the zones of growth inhibition are directly proportional to the log10 of the penicillin concentration Bioassay of Penicillin Penicillin 15 μg/ml Penicillin 0.12 μg/ml Penicillin 3 μg/ml Penicillin 0.6 μg/ml From a standard curve the concentration of an unknown penicillin solution can be determined by measuring the zone diameter of growth inhibition Bioassay of Penicillin 40 Diameter of Zone of Inhibition (mm) 35 30 25 20 15 10 5 0 0.1 1 10 Log10 Penicillin Concentration (μg/ml) 100 Multi-resistant MRSA This disc sensitivity test demonstrates that the methicillinresistant S. aureus [MRSA] strain shown is resistant to 11 of the 12 antimicrobial agents tested – the exception (arrowed is vancomycin, a glycopeptide antibiotic commonly used to treat MRSA infections in hospitals
