Staphylococcus Spp

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Staphylococcus Zari Esa M.SalehMSc. Medical Microbiology Family: Micrococcaceae Genus:

Staphylococcus Staphylococcus aureus. Staphylococcus epidermidis. Staphylococcus saprophyticus.

MicrococcusTaxonomy 2Genus Staphylococcus Staphylococci comprises of around 35-40 species, however not all of the members cause disease.

Many of the members of the staphylococcus genera are considered to be normal flora on the skin and mucus membranes.

Staphylococci are the most frequent bacteria associated with the production of pus.

3 Gram stain Staphylococci are Gram-positive, spherical cells (0.5-1.5 mm) cocci. Arranged in single, pairs, tetrad and irregular clusters. The grapelike orientation is the most common arrangement associated with the staphylococci. Staphylococci are non-motile, non-spore forming, non-capsulated bacteria. Microscopical characteristicsGram-stained smear of Staphylococci

Mixture of Gram-positive cocci (Staphylococcus aureus) and Gram-negative rods (Escherichia coli).

Direct gram-stained of Staphylococci from sputum sample.

Gram-positive cocci seen intracellularlly (within polymorphonuclear leukocyte).Irregularly clustered Gram-positive cocci (staphylococcus aureus) in and outside neutrophils.

Staphylococcus are aerobic and facultative an-aerobic, grow well on simple media. Colonies are 2mm in diameter after 24h of incubation at 37 C. Colonies appear convex, circular opaque, smooth, moist with an entire edge and pigmented.Staphylococcus are tolerant to salt (7-10% Nacl)

Cultural CharacteristicsNutrient agar- the colonies are large circular convex, smooth, shiny, opaque .They are easily emulsifiable.Most stains produce golden pigmentation.

Cultural Characteristics

10Blood agar-colonies are white to creamy.Most stains produce Beta-haemoloysis.Cultural Characteristics

Mannitol salt agar (MSA)-Staphylococcus aureus ferments mannitol and appear as yellow colonies.MSA is a useful selective medium for recovering Staphylococcus aureus from faecal specimens, when investigating food poisoning.Cultural Characteristics

12Cultural Characteristics

S. aureus (ferment)MSA Acid (yellow) Non-S. aureus (No fermentation) MSANo acid (Red)Biochemical testsCatalase test - catalase is an enzyme that removes hydrogen peroxide (H2O2) by catalyzing the breakdown of the molecules into water and oxygen. The enzyme is produced by all species of Staphylococcus, so they can be differentiated from Streptococci, which can not produce the enzyme.Procedure. Take a colony of the organism to be tested and place on a glass slideAdd a drop of 3% hydrogen peroxide onto the colony. The immediate formation of oxygen bubbles is evidence that the organism produces catalase (positive reaction).Few or no bubbles after 20 seconds indicates a negative reaction.

Biochemical tests

With respect to the blood agar, blood agar can interfere with catalase test since blood cells are catalase positive.Any contaminating agar could give a false positive result for the catalase test.Therefore it is recommended to use colonies that are on another agar.DO NOT use a metal loop or needle with H2O2, it will give a false positive and degrade the metal.Biochemical testsCoagulase test this test is used to differentiate Staphylococcus aureus(positive) from Coagulase Negative Staphylococcus (CONS). Coagulase is an enzyme produced by staphylococcus aureus, which converts (soluble ) fibrinogen in plasma to (insoluble) fibrin. Staphylococcus aureusproduces two forms of coagulase, bound and free.Slide coagulase test is done to detect bound coagulase or clumping factor.Clumping factor is a protein located on the surface of Staphylococcus aureus cells.

Biochemical tests17Clumping factor causes agglutination in human, rabbit, or pig plasma. This factor converts fibrinogen to fibrin, resulting in agglutination.Procedure. Place a drop of sterile water on a slide, then emulsify a colony to be tested.Add a drop of rabbit plasma, and rock the slide gently for 5 to 10 seconds and look for clumping (coagulation)

Biochemical testsAgglutination is interpreted as a positive reaction and identifies the organism as S. aureus. No agglutination is interpreted as a negative reaction. Biochemical tests

19Tube coagulase test is done to detect free coagulase.The test detects staphylocoagulase enzyme, which is also know as free coagulase. The enzyme is not bound to the surface of the cells. Staphylocoagulase is an extracellular molecule that causes a clot to form when bacterial cells are incubated with plasma.Biochemical testsProcedure. Emulsify several isolated colonies of test organism in 0.5 ml of rabbit plasma to give a milky suspension.Incubate tube at 35oC for 4 hours.The tube should be checked every 30 minutes for up to 4 hours to see if a clot has formed. Formation of a clot indicates a positive reactionBiochemical tests

Serum can not be used because it lacks the clotting factors found in the plasma, including fibrinogen.The slide test should not be performed with organisms taken from high-salt media such as Mannitol Salt Agar, as the salt content can create false positives.Some staphylococcus aureus produce staphylokinase enzyme along with staphylocoagulase enzyme.Staphylokinase is an enzyme that can dissolve clots.Therefore if we waited more than 4 hours to check the tube, the test result would be negative when in fact a clot could have been there previously, but now it has been dissolved. This is why we should check every 30 minutes.

Biochemical testsMicrococcus Micrococcus is another genera found in the Micrococcaceae family.Micrococcus species are found in the environment and are indigenous skin flora. Because of this, they are usually considered contaminates when isolated from clinical specimens. Micrococcus is catalase positive and coagulase negative.

One way to distinguish them from the staphylococcus is its yellow appearance on colony morphology.Microdase disc can also be used to differentiate between staphylococcus and micrococcus based on a color change.

Micrococcus Microdase (Modified Oxidase) Test- is a rapid test, which detect the cytochrome enzyme.All microccocci possess cytochrome C enzyme , whereas most staphylococci lack it.Micrococcus species give a blue colour, so a positive result for cytochrome C.Staphylococcus species give no colour and therefore is negative for cytochrome C.Micrococcus

Positive Negative Summary Micrococcaceae

26Staph. aureusStaph. EpidermidisStaph. saprophyticusMicrococcus

Colony MorphologyOpaque, smooth, raised, entire, white-golden(cream)Opaque, smooth, raised, entire, gray-whiteOpaque, smooth, raised, entire, butyrous, glossy, white-yellowOpaque, smooth, raised, white, bright yellow

HemolysisMost are beta hemolyticNon-hemolyticNon-hemolyticNon-hemolytic

Gram morphologyGPC in clusters, pairs, short chains or singlyGPC in clusters, pairs, short chains or singlyGPC in clusters, pairs, short chains or singlyGPC in pairs and tetrads

CatalasePosPosPosPos

Glucose fermentationFermenterFermenterFermenterOxidizer

Modified OxidaseNegNegNegPos

Coagulase Production (tube)PosNegNegN/A

Clumping factor (slide or latex Coagulase test)PosNegNegNeg