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MLAB 2434: MICROBIOLOGY KERI BROPHY-MARTINEZ Staphylococci As with most infections, the development of staphylococcal infection is determined by the virulence of the ... – PowerPoint PPT presentation

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Title: MLAB 2434: Microbiology Keri Brophy-Martinez


1
MLAB 2434 MicrobiologyKeri Brophy-Martinez
  • Staphylococci

2
Taxonomy
  • Family Micrococcaceae
  • Genus
  • Staphylococcus
  • Coagulase positive
  • Coagulase negative
  • Micrococcus

3
Genus Staphylococcus
  • Coagulase positive
  • Coagulase negative
  • S. aureus
  • S. epidermidis
  • S. saprophyticus
  • S. haemolyticus

4
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
5
Staphylococcus General Characteristics
  • Nonmotile
  • Nonspore-forming
  • Nonencapsulated
  • Catalase-producing
  • Oxidase negative
  • Glucose fermenters
  • Primarily aerobic, some facultatively anaerobic

6
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

7
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

8
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

9
Staphylococcus aureus
  • Infection will elaborate inflammatory response
    with GPC accumulating as pus
  • Pus mix of active and inactive neutrophils,
    bacterial cells and extravascular fluid

10
Virulence Factors of S. aureus
  • Enterotoxins
  • Cytolytic toxins
  • Enzymes
  • Protein A

11
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

12
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

13
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.

14
Virulence Factors Extracellular Enzymes (cont)
  • Penicillinase
  • Confers resistance
  • DNase
  • Degrades DNA
  • Beta-lactamase
  • Cuts the beta lactam wall of certain antibiotics

15
Virulence Factors Protein A
  • Protein A
  • Found in cell wall
  • Binds to Fc part of IgG
  • Blocks phagocytosis

16
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
17
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

18
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.

19
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

20
Coagulase-Negative Staphylococci
  • Habitat
  • Skin and mucous membranes
  • Common human isolates
  • S. epidermidis
  • S. saprophyticus
  • S. haemolyticus

21
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

22
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

23
Coagulase-Negative Staphylococci Staphylococcus
Haemolyticus
  • Habitat skin and mucous membranes
  • Rarely implicated in infections
  • Associated with wound infections, bacteremia,
    and endocarditis

24
Break Time!!!
25
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

26
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
27
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
28
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

29
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

30
Catalase Test Interpretation
  • Presence of bubbles
  • Positive
  • Staphylococci
  • Absence of bubbles
  • Negative
  • Streptococci

31
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

32
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

33
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

34
Novobiocin Susceptibility Test
  • Test to differentiate coagulase-negative
    staphylococci from S.saprophyticus from urine
    samples
  • S. saprophyticus is resistant (top)
  • Other CNS are susceptible

35
Micrococcus
  • Rarely produces disease
  • Found in environment and indigenous skin flora
  • Catalase
  • Coagulase
  • Produces yellow pigment
  • Microdase disc differentiate between Staph
    Micrococcus

36
Schematic Diagram for Identifying Staphylococcal
Species
37
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

38
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

39
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

40
Vancomycin-resistant staphylococci
  • VRSA vancomycin resistant Staphylococcus aureus
  • VISA vancomycin intermediate Saphylococcus
    aureus
  • Detection
  • Vancomycin screening media

41
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

42
Summary Micrococcaceae
43
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.
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