Module 6.1: Staphylococcus And Micrococcus

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•Natural habitants of the skin and mucus membranes in humans

•Can be ubiquitous in the environment

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GRAM POSITIVE COCCI

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•Peptococcus •Peptostreptococcus •Sarcina

•Peptococcus •Peptostreptococcus •Sarcina

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ANAEROBES

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•Micrococcus •Staphylococcus •Streptococcus

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AEROBES

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Skin and soft tissue infections, bacteremia, toxic shock syndrome

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Staphylococcus aureus

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All cocci are Gram-positive except

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NVB

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Pharyngitis, skin and soft tissue infections

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Streptococcus pyogenes

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Neonatal bacteremia and meningitis

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Streptococcus agalactiae

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CAP, meningitis

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Streptococcus pneumoniae

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Nosocomial UTIs and bacteremia

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Enterococcus faecalis and E. faecium

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BAP: β hemolytic; round, smooth, opaque and butyrous; gray to deep golden yellow MSA: yellow colonies

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Staphylococcus aureus

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T/F:

1st: Greek word which means bunches of grapes is kokos

2nd: Greek word which means berries is staphule

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FALSE: Staphule

FALSE: Kokos

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Skin infections are: impetigo, boils, folliculitis, mastitis, and infection of surgical wounds are clinical manifestations of what?

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S. aureus

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Binds to Fc portion of IgG; inhibits phagocytosis; involved in coagglutination

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Protein A

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aka clumping factor; converts fibrinogen to fibrin; coats PMN with fibrin to inhibit phagocytosis

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Coagulase

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Also fibrinolysin; enables infection once fibrin clot is dissolved

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Staphylokinase

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Aka epidermolytic toxin; responsible for scalded skin syndrome (SSS) aka Ritter- Lyell disease

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Exfoliatin

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Hydrolyze lipids in plasma and skin; responsible for skin infections

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Lipase

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Spreading factor: dissolves hyaluronic acid in connective tissues

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Hyaluronidase

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aka Panton-Valentine leukocidin (PVL) Lyse neutrophils and macrophages

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Leukocidin

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Lyse RBCs Causes anemia and makes iron available for bacteria

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Hemolysin

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Food poisoning (preformed toxin) occurs 1-6 hours after ingestion

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Enterotoxins A–E

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Aka Enterotoxin F Disease In women who use tampons

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Toxic shock syndrome toxin (TSST-1)

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S. aureus strain resistant to antibiotics such as

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methicillin, nafcillin, and oxacillin

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•Acquired after prolonged stay in the hospital, close contact with carriers, broad-spectrum antibiotics • In the community, mostly skin infections

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Methicillin-resistant S. aureus (MRSA)

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changes in the color of MRSA colonies within 24-48 hours using CHROM agar against colorless colonies of non-MRSA

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(+) Chromogenic test

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CoNS (Non–Staphylococcus aureus)

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Coagulase- Negative Staphylococci

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NOVOBIOCIN SUSCEPTIBLE

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S. epidermidis, S. capitis, S. haemolyticus, S. hominis subsp. hominis, S.lugdunensis, S. saccharolyticus, S. warneri, etc.

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NOVOBIOCIN RESISTANT

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S. cohnii, S. kloosii, S. saprophyticus, and S. xylosus

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Very similar to S. aureus

Gram-positive cocci in clusters

BAP: small to medium; opaque, gray-white colonies; most colonies nonhemolytic

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Staphylococcus saprophyticus

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•Common cause of UTI in young sexually active women

•Normal Flora of skin and urethra

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Staphylococcus saprophyticus

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Biochemical Characteristics

•Resistance to novobiocin

•Negative phosphatase reaction

•Resistant to nalidixic acid

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Staphylococcus saprophyticus

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1.Micrococcus

2. Arthrobacteria

3. Kocuria

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Family Micrococcaceae

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•Occur worldwide and are ubiquitous

•Indigenous skin biota, often recovered with staphylococci

•Rarely infectious

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MICROCOCCUS

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Gram-positive cocci typically in tetrads but may also appear in pairs or clusters

BAP: Small to medium; opaque, convex; nonhemolytic; wide variety of pigments (white, tan, yellow, orange, pink)

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Micrococcus spp.

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Grows on 5% NaCl but not on 7.5% NaCl

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Micrococcus spp.

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(+)Catalase

(-) Coagulase and Nitrate

(+) Microdase Susceptible to Novobiocin and Bacitracin

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Biochemical Characteristics of Micrococcus spp.

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T/F:

1st: Staphylococci are Gram-negative

2nd: Staphylococcus – grape-like

3rd: Micrococcus – tetrads 4th: Sarcinae – cuboidal

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Statement 2nd, 3rd and 4th are True

1st: (Gram-positive)

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•Sheep BA recommended for 1°isolation

•Staph: creamy white pinhead colonies

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Blood agar plate (BAP)

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•S. aureus: jet-black colonies

•Other staph: inhibited or gray colonies

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Tellurite Lysine Agar

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Used for heavily contaminated specimens

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Mannitol Salt Agar (MSA)

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•Yellow colonies

•S. aureus

•S. saphrophyticus

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Mannitol fermenters

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•Pink colonies

•S. epidermidis

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Mannitol nonfermenters

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•pH indicator: phenol red

•High salt concentration: 7.5-10% NaCl

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Mannitol Salt Agar (MSA)

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used for purulent exudates

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Colistin-nalidixic acid (CNA)

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used for heavily contaminated specimens

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Phenylethyl alcohol (PEA)

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•Differentiates Staphylococcus (+) from Streptococcus (-)

•Reagent: 3% H2O2

•(+) result: effervescence

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Catalase test

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Staphylococcus aureus

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Positive in Catalase test

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Streptococcus pyogenes

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Negative in Catalase test

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T/F:

Catalase is enzyme that decomposes hydrogen peroxide (H2O2) to water and ground-state oxygen.

H2O2 + H2O2 Catalase 2H2O + O

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TRUE

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Most useful single criterion for ID of S. aureus

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Coagulase Test

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S. aureus (ATCC25923)

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Positive in Coagulase Test

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S. epidermidis (ATC12228)

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Negative in Coagulase Test

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Reagent of Coagulase Test

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Rabbit Plasma

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Why is Citrate not used in Coagulase Test?

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It's False-Positive

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•Rapid screening test

•Detects cell bound coagulase

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Slide Coagulase

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•Confirmatory for (-) slide method •Detects free coagulase

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Tube Coagulase

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Other slide coagulase- positive Staphylococcus:

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• S. lugdunensis

•S. schleiferi

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What is Colony + plasma, rock slide gently for 5-10 seconds for?

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Procedure for Slide Coagulase

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clot or coagulum formation within 30 seconds

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POSITIVE CONTROL FOR SLIDE COAGULASE

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colony + saline/distilled water

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Negative Control for Slide Coagulase

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Colonies transferred into a tube with plasma and incubated at 35°C for 4 hours

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Tube Coagulase

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gel-like fibrin clot of any size

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Positive Result for Tube Coagulase

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T/F:

• Checked every 30 minutes for 4 hours @ 35°C

• If (-) for 4 hours incubate for another 24 hours @RT

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True-True

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Other tube coagulase- positive Staphylococcus:

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S. hyicus, S. intermedius, S. lutrae, S. delphini, and S. schleiferi subsp. coagulans

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Determines if an organism can produce DNAse (hydrolyzes DNA)

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DNAse Test

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What is the reagent of DNAse?

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DNAse agar with methyl green/ toluidine blue as indicator

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Positive Result of DNAse?

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clearing of media (methyl green)/ blue into rose pink (toluidine blue)

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T/F:

Are positive in DNAse Test S. aureus, Moraxella, Serratia?

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True

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Resistant/Susceptible Novobiocin Susceptibility Test:

•S. epidermidis

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Susceptible

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Resistant/Susceptible in Novobiocin Susceptibility Test:

•S. saprophyticus

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Resistant

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T/F:

•Reagent of Novobiocin is 5 ug Novobiocin and sheep BAP

•Positive result: ZOI >16mm

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True-True