Staphylococcus

What are characteristics of staphylococcus

• gram positive cocci

• forms tetrads/clusters

• non-motile

• facultative anaerobes

What are the Steps in Gram Staining?

1. Crystal Violet

   1. both turn purple

2. Iodine

   1. fixes purple onto specimen

3. alcohol (decolorization)

   1. removes color from gram-negative

4. safarin (counterstain)

   1. dyes gram negative bacteria pink

Catalase Test

• all facultative anaerobes are catalase +

• 1 drop of hydrogen peroxide is added to slide with specimen

• bubble formation = positive result

Micrococcus luteus

• skin contaminant, lives on skin

• not a pathogen

• can contaminate specimen

• cultures grown have a yellow appearance

Results that would indicate Staphylococcus

Lysostaphin: susceptible

Ferment Glucose: +

Cytochrome C Oxidase : -

Bacitracin sensitivity: resistant

Results that would indicate Micrococcus

Lysostaphin: resistant

Ferment Glucose: -

Cytochrome C Oxidase : +

Bacitracin sensitivity: susceptible

A positive modified oxidase test would indicate what specimen?

• Micrococcus species

What is the most clinically significant species of staphylococci?

• Staphylococcus aureus

  • important causes of healthcare-associated infections

Where does staphylococci inhabit?

• skin and mucous membranes of humans and animals

• normal flora of:

  • anterior nares

  • oropharynx

  • skin

  • genitourinary tract

What are different virulence factors associated with S. aureus

1. Protein A

2. Exotoxins

3. Leukocidins

4. Capsule

5. Coagulases

6. Glycocalyx

Protein A

• located on staphylococcus

• binds to the Fc portion of IgG

• blocks opsonization, prevents phagocytosis, and impairs complement activation

What exotoxin causes food poisening?

• enterotoxins

What are different examples of exotoxins?

• hemolysins

• exfoliants

• enterotoxins

• toxic shock syndrome toxin 1

Toxic Shock Syndrome Toxin 1 (TSST-1)

• superantigen

• stimulates T-cell proliferation and cytokine release

• leads to systemic effects of toxic shock

Exfoliatins

• protease activity causes epidermal layer of skin to slough off

• can cause staphylococcal scalded skin syndrome

Leukocidins

• kills leukocytes

• ex: Panton-Valentine leukocidin

Capsule

• polysaccaride

• protects from phagocytosis

Coagulases

• bound to surface or secreted

  • bound: clumping factor

  • free: staphylocoagulase

• convert fibrinogen to fibrin to form a clot

Glycocalyx

• thick layer over organism made of sugar

  • ex Biofilms: protec from antibiotics/immune system

What people are at high risk for staphylococcus infections

• diabetics

• immunocompromised/suppressed

• burn patients

• neomates

• patients in the ICU

Furuncles

• boils

• obstructed oil gland or hair follicle

• type of deep folliculitis

• influx of neutrophils to the site of infection causes boil formation

Carbuncles

• deeper abscess, can spread from multiple furuncles (cluster of boils)

Impetigo

• common in children

• contagious type

  • 70%, seen in face (nose/mouth) common in children

• Bullous type

  • 30%, >5 mm blisters, primarily neonates and younger children, seen on trunk or extremities

Cellulitis

• infection of connective tissue (arms/ legs)

• deeper infection

• affects dermis and subcutaneous fat

Staphylococcal scaled skin syndrome (SSSS)

• caused by exfoliatins

  • leads to epidermal detachment

• profuse skin peeling

• due to immature renal system being unable to filter out the toxin

• seen in newborns and young children

Toxic Shock Syndrome

• caused by toxic shock syndrome toxin 1 and staphylococcal enterotoxin B

• highly absorbent tampons

  • TSST1 is absorbed through vaginal mucosa

• non-menstrual form

  • postsurgical infections, skin scrape, wound

Endocarditis

• inflammation of endocardium

• results in damage to cardiac structures

• can lead to death in weeks if not treated if it is acute

• subacute is a slower disease

What conditions can arise from staphylococcos diseases?

• furuncles

• carbuncles

• impetigo

• cellulitis

• Staphylococcal scaled skin syndrome

• toxic shock syndrome

• endocarditis

• food poisoning

• osteomyelitis, pneumonia, sepsis, septic arthritis, UTIs

Food Poisoning cause

• Intoxication: enterotoxins

• resolves within 24 hours

What is not helpful when diagnosing staphylococcus diseases?

• serology

What do DNA probes loo for when trying to diagnose a staphylococcus disease?

• genes for SEA, SEB,SEC, and TSST-1

  • specific for enterotoxins

• probes are not readily available

What culture media is typically used to staphylococcus isolation and identification?

• sheep blood agar (SBA)

• Staphylococcus aureus will have beta hemolysis (clearing around colonies)

Manitol Salt Agar (MSA)

• has 7.5-10% sodium chloride to inhibit growth of other bacteria

• tests for mannitol fermentation

Staphylococcus aureus vs Staphylococcus epidermis on MSA Plate

Staphylococcus aureus

• growth

• mannitol fermentation results in yellow color

vs Staphylococcus epidermidis

• growth

• no mannitol fermentation

Coagulase test results

• clot formation = positive = Staphylococcus aureus

• no clot formation = negative

Agglutination Test for clumping factor: slide coagulase

• clumping = positive = Staphylococcus aureus

• no clumping = negative

Rapid Staph Latex

• looks for clumping factor and protein A

  • both components of Staph. a

Small Colony Variants (SCV)

• nonhemolytic

• grow slowly

• some are thymidine dependent

  • will not grow unless thymidine is in plate medium

Treatment for Staphylococcal diseases

• almost always beta-lactamase positive

  • penicillinases

• oxacillin, clindamycin, trimethoprim sulfamethoxazole

• vancomycin

Methicillin-resistant Staphylococcus aureus (MRSA)

• have the mecA gene which results in altered penicillin binding protein

• beta-lactan antibiotics can NOT be used to treat MRSA

What phenotypic tests are used to diagnose MRSA?

• cefoxitin

• oxacillin

What genotypic testing is used to diagnose MRSA?

• mecA detection via PCR

What culture medium is specifically used to look for MRSA?

• MRSA select (Bio-rad)

• CHROMagar MRSA remel

Staphylococcus epidermidis

• most common coagulase negative staphylococcus

• grey colonies and no beta hemolysis

Vancomycin-intermediate Staphylococcus aureus (VISA)

• usually different colony morphology compared to other S. aureus strains

• small colony variants

• vancomycin can NOT be used to treat infection

Vancomycin-resistant Staphylococcus aureus (VRSA)

• very rare, acquired from enterococci

• vancomycin can NOT be used to treat infection

Pathogenesis of Staphylococcus Epidermidis

• HAIs (catheters or implants)]

• associated with foreign body implant infections

  • biofilms - heart, knees, hips

• bacteremia, UTIs, osteomyelitis

Types of Staphylococcus epidermidis

• S. haemolyticus

• S. hominis

• S. simulans

S. saprophyticus

• 2nd most common cause of commonly acquired UTIs in female adolescents

• white, non hemolytic colonies

S. haemolyticus

• second most commonly isolated CoNS

S. lugdunensis

• pathogenesis: endocarditis

• mimics S. aureus

How do you identify coagulase negative staphylococci (CoNS)

• look for non-hemolysis

• Novobiocin disk sensitivity

  • S. saprophyticus is novobiocin resistant

• biochemical testing

  • urease, carbohydrates,

S. aureus

• Tube Coagulase: +

• Clumping Factor: +

• PYR: -

• ODC: -

S. epidermidis

• Tube Coagulase: -

• Clumping Factor: -

• PYR: —

• ODC: +

S. haemolytiucus

• Tube Coagulase: -

• Clumping Factor: -

• PYR: +

• ODC: —

S. lugdunensis

• Tube Coagulase: -

• Clumping Factor: +

• PYR: +

• ODC: +

S. saprophyticus

• Tube Coagulase: -

• Clumping Factor: -

• PYR: -

• ODC: -

how to determine S. aureus or CoNS

• do a colony morphology and gram stain

• catalase test

• coagulase

  • positive: S. aureus

  • negative: CoNS

What test will definitively identify staphylococci?

• MALDI-TOF analysis