MLAB 2434: Microbiology Keri Brophy-Martinez

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MLAB 2434 MicrobiologyKeri Brophy-Martinez

• Staphylococci

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Taxonomy

• Family Micrococcaceae
• Genus
• Staphylococcus
• Coagulase positive
• Coagulase negative
• Micrococcus

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Genus Staphylococcus

• Coagulase positive

• Coagulase negative

• S. aureus

• S. epidermidis
• S. saprophyticus
• S. haemolyticus

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Staphylococcus Gram Reaction and Morphology

• Gram-positive spherical cells (0.5-1.5 mm) in
  singles, pairs, and clusters
• Appear as bunches of grapes

Gram-stained smear of staphylococci from colony
Scanning electron micrograph of staphylococci
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Staphylococcus General Characteristics

• Nonmotile
• Nonspore-forming
• Nonencapsulated
• Catalase-producing
• Oxidase negative
• Glucose fermenters
• Primarily aerobic, some facultatively anaerobic

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StaphylococcusGeneral Characteristics (cont)

• Bacitracin resistant
• Grow on agar that contains peptone
• Inhibited by media that has high bile salt
  concentration
• Some are ß-hemolytic
• Colony morphology buttery looking, cream or
  white colored

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Staphylococcus aureus

• Primary pathogen of the genus
• Habitat
• Anterior nares (carriers)
• Colonization axilla, vagina, pharynx
• Produce superficial to systemic infections
• Skin
• Bacterial sepsis
• Hospital acquired infections

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Staphylococcus aureus

• Mode of transmission
• Traumatic introduction
• Direct contact with infected person
• Inanimate objects
• Predisposing conditions
• Chronic infections
• Indwelling devices
• Skin injuries
• Immune response defects

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Staphylococcus aureus

• Infection will elaborate inflammatory response
  with GPC accumulating as pus
• Pus mix of active and inactive neutrophils,
  bacterial cells and extravascular fluid

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Virulence Factors of S. aureus

• Enterotoxins
• Cytolytic toxins
• Enzymes
• Protein A

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Virulence Factors Enterotoxins

• Enterotoxins
• Heat-stable exotoxins that cause diarrhea and
  vomiting
• Exotoxin protein produced by a bacteria and
  released into environment
• Heat stable _at_ 100o C for 30 minutes
• Implications
• Food poisoning
• Toxic shock syndrome
• Pseudomembranous enterocolitis

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Types of enterotoxins

• Exfoliatin
• Epidermolytic toxin
• TSST-1 Toxic shock syndrome toxin-1
• Multisystem disease
• Stimulates T cell production cytokines
• Cytolytic Toxins
• Affects RBCs and WBCs
• Hemolytic toxins alpha, beta, gamma, delta
• Panton-Valentine leukocin, lethal to WBCs

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Virulence Factors Extracellular Enzymes

• Hyaluronidase
• Hydrolyzes hyaluronic acid in connective tissue
  allowing spread of infection
• Staphylokinase
• Fibrinolysin which allows spread of infection
• Coagulase
• Virulence marker
• Lipase
• Allows colonization by acting on lipids present
  on the surface of the skin.

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Virulence Factors Extracellular Enzymes (cont)

• Penicillinase
• Confers resistance
• DNase
• Degrades DNA
• Beta-lactamase
• Cuts the beta lactam wall of certain antibiotics

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Virulence Factors Protein A

• Protein A
• Found in cell wall
• Binds to Fc part of IgG
• Blocks phagocytosis

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Staphylococcus aureus Clinical Infections

• Skin and wound
• Impetigo
• Furuncles/Boils (Infection of hair follicles
  usually in areas that sweat)
• Carbuncles (clusters of boils)
• Surgical wound infections

Bullous impetigo
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Staphylococcus aureus Clinical Infections
(cont)

• Skin and wound
• Scalded skin syndrome Ritters disease
• Extensive exfoliative dermatitis
• Young children and newborns
• Toxic Shock Syndrome
• Multisystem disease
• Caused by TSST-1
• Affects women, men, and children

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Staphylococcus aureus Clinical Infections

• Food poisoning
• Source is infected food handler
• Enterotoxin A the most common cause
• Foods affected include meat, dairy products,
  bakery goods with cream fillings, and salads made
  with eggs and mayonnaise.

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Coagulase-Negative Staphylococci

• Found as indigenous flora
• Presence can indicate contamination
• Seeing an increase due to prosthetic devices,
  catheters and immunocompromised
• Abbreviated CNS or CoNS

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Coagulase-Negative Staphylococci

• Habitat
• Skin and mucous membranes
• Common human isolates
• S. epidermidis
• S. saprophyticus
• S. haemolyticus

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Coagulase-Negative Staphylococci Staphylococcus
epidermidis

• Predominantly hospital acquired infections
• Skin flora gets introduced by catheters, heart
  valves, CSF shunts
• Produces a slime layer that helps adherence to
  prosthetics and avoidance of phagocytosis
• UTIs are a common result

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Coagulase-Negative Staphylococci Staphylococcus
saprophyticus

• UTIs in young sexually active women
• Due in part to increased adherence to epithelial
  cells lining the urogenital tract
• Rarely present in other skin areas or mucous
  membranes
• Urine cultures
• If present in low amounts, it is still considered
  significant

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Coagulase-Negative Staphylococci Staphylococcus
Haemolyticus

• Habitat skin and mucous membranes
• Rarely implicated in infections
• Associated with wound infections, bacteremia,
  and endocarditis

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Break Time!!!
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Laboratory Diagnosis Specimen Collection and
Handling

• Samples must be taken from the actual site of
  infection
• Prevent delay in transport of collected material
  from infected sites
• Transport in appropriate collection device that
  would prevent drying and minimize growth of
  contaminating organisms

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Laboratory Diagnosis Direct Smear Examination

• Microscopic Examination
• Gram reaction
• Gram-positive cocci
• Cell arrangement
• Pairs and clusters
• Presence/Absence of PMNs
• Numerous polymorphonuclear cells (PMNs)

Insert Figure 10-1
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Laboratory Diagnosis Cultural Characteristics-

• Staphylococcus aureus
• Colony morphology
• Smooth, butyrous, white to yellow, creamy
• Grow well _at_ 18-24 hours
• S. aureus may produce hemolysis on blood agar

S. aureus
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Laboratory Diagnosis Cultural Characteristics

• S. epidermidis
• Smooth, creamy, white
• Small-to medium- sized, usually non-hemolytic
• S. saprophyticus
• Smooth, creamy, may produce a yellow pigment

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Identification Tests Catalase

• Principle tests for enzyme catalase
• 2 H2O2 2 H2O O2
• Procedure
• Smear a colony of the organism to a slide
• Drop H2O2 onto smear
• Observe

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Catalase Test Interpretation

• Presence of bubbles
• Positive
• Staphylococci
• Absence of bubbles
• Negative
• Streptococci

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Identification test slide coagulase test

• Differentiates members within the Staphylococci
• Detects clumping factor found in S. aureus
• Procedure
• Place a drop of sterile water on a slide and
  emulsify a colony
• Add a drop of rabbit plasma to the suspension
• Observe
• Agglutination Positive
• No agglutination Negative

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Identification Tests Coagulase Test

• Detects the extracellular enzyme free
  coagulase or staphylocoagulase
• Causes a clot to form when bacterial cells are
  incubated with plasma
• Procedure
• Inoculate rabbit plasma with organism and
  incubate at 35-37 0 C
• Observe at 30 minutes for the presence of a clot
• Continue for up to 24 hours, if needed

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Identification Tests Rapid Coagulase Test

• Latex Agglutination Assays
• Detects cell-bound clumping factor, protein A
  or a combination of both
• Procedure
• Varies depending on kit type
• Positive reaction demonstrated by agglutination

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Novobiocin Susceptibility Test

• Test to differentiate coagulase-negative
  staphylococci from S.saprophyticus from urine
  samples
• S. saprophyticus is resistant (top)
• Other CNS are susceptible

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Micrococcus

• Rarely produces disease
• Found in environment and indigenous skin flora
• Catalase
• Coagulase
• Produces yellow pigment
• Microdase disc differentiate between Staph
  Micrococcus

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Schematic Diagram for Identifying Staphylococcal
Species
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Antimicrobial Susceptibility

• For nonbeta-lactamase producing S. aureus
• Use pencillin
• Penicillinase-resistant synthetic penicillins
  (methicillin, nafcillin, oxacillin,
  dicloxacillin)
• Beta-lactamase producers break down the
  beta-lactam ring of penicillin so it inactivates
  antibiotic before it acts on bacterial cells

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Methicillin-Resistant Staphylococci

• MRSA
• Methicillin-resistant S. epidermidis
• MRSE
• Infection control
• Barrier protection
• Contact isolation
• Handwashing
• Treat with vancomycin
• Test for susceptibility with cefoxitin disk

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Methicillin-Resistant Staphylococci (Contd)

• mecA gene
• Encodes penicillin-binding proteins (PBPs)
• Causes drug ineffectiveness
• Gold standard
• Nucleic acid probe or PCR for the mec A gene

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Vancomycin-resistant staphylococci

• VRSA vancomycin resistant Staphylococcus aureus
  
• VISA vancomycin intermediate Saphylococcus
  aureus
• Detection
• Vancomycin screening media

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Antimicrobial Susceptibility

• Macrolide Resistance
• Clindamycin sensitivity often requested by
  physician to treat Staph skin infection.
  Referred to as D test
• Clindamycin resistance is often inducible meaning
  it only is detectable when bacteria are also
  exposed to erythromycin

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Summary Micrococcaceae
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References

• Engelkirk, P., Duben-Engelkirk, J. (2008).
  Laboratory Diagnosis of Infectious Diseases
  Essentials of Diagnostic Microbiology .
  Baltimore, MD Lippincott Williams and Wilkins.
• http//archive.microbelibrary.org/ASMOnly/Details.
  asp?ID2037
• http//brawlinthefamily.keenspot.com/gallery/2009-
  10-18-breaktime/
• http//ericaandkevin.pbworks.com/w/page/5827086/Gr
  am-Stain-and-Other-Tests
• http//faculty.matcmadison.edu/mljensen/111CourseD
  ocs/111Review/Unit2Reviews/micrococcaceae_answers.
  htm
• http//jeeves.mmg.uci.edu/immunology/Assays/LatexA
  gglut.htm
• Mahon, C. R., Lehman, D. C., Manuselis, G.
  (2011). Textbook of Diagnostic Microbiology (4th
  ed.). Maryland Heights, MO Saunders.