Staphylococci &

Micrococci
Learning outcomes:

At the end of this class, you all should be able to:

1.Describe the:
 cellular morphology
 expected staining reaction
 optimum atmosphere
 temperature and growth requirements
 colonial morphology
 pathogenesis of disease state
 and the main differentiating feature of the major bacterial
Taxonomy

Family: Micrococcaceae
Genus:
Staphylococcus
Coagulase positive
Coagulase negative
Micrococcus
Genus Staphylococcus

Coagulase positive Coagulase negative

 S. aureus  S. epidermidis
 S. saprophyticus
 S. haemolyticus
 Staphylococcus:
Gram Reaction and Morphology

 Gram-positive spherical cells (0.5-

1.5 m) in singles, pairs, and
clusters
 Appear as “bunches of grapes”

Gram-stained smear of
staphylococci from colony

Scanning electron micrograph of

staphylococci
 Staphylococcus:
General Characteristics

 Nonmotile

 Non–spore-forming

 Nonencapsulated

 Catalase-producing

 Oxidase: negative

 Glucose fermenters

 Primarily aerobic, some facultatively anaerobic

Staphylococcus:
General Characteristics (con’t)
 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
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
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
 Staphylococcus aureus

 Infection will elaborate inflammatory response with GPC

accumulating as pus
 Pus: mix of active and inactive neutrophils, bacterial cells and
extravascular fluid
Virulence Factors of S.
aureus

 Enterotoxins
 Cytolytic toxins
 Enzymes
 Protein A
Virulence Factors: Enterotoxins

 Enterotoxins:
 Heat-stable exotoxins that cause diarrhea and vomiting
 Exotoxin: protein produced by a bacteria and released into environment
 Heat stable @ 100o C for 30 minutes

 Implications
 Food poisoning
 Toxic shock syndrome
 Pseudomembranous enterocolitis
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
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.
Virulence Factors:
Extracellular Enzymes
(con’t)

 Penicillinase:
 Confers resistance
 DNase:
 Degrades DNA

 Beta-lactamase:
 Cuts the beta lactam wall of certain antibiotics
Virulence Factors: Protein A

Protein A:
Found in cell wall
Binds to Fc part of IgG
Blocks phagocytosis
 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
Staphylococcus aureus:
Clinical Infections (con’t)

 Skin and wound

 Scalded skin syndrome= Ritter’s disease
Extensive exfoliative dermatitis
Young children and newborns

 Toxic Shock Syndrome

Multisystem disease
Caused by TSST-1
Affects women, men, and children
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.
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
Coagulase-Negative
Staphylococci

 Habitat:
Skin and mucous membranes

 Common human isolates

S. epidermidis
S. saprophyticus
S. haemolyticus
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
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
Coagulase-Negative
Staphylococci: Staphylococcus
Haemolyticus

 Habitat: skin and mucous membranes

 Rarely implicated in infections

 Associated with wound infections, bacteremia, and

endocarditis
Break Time!!!
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
 Laboratory Diagnosis:
Direct Smear Examination

Microscopic Examination
o Gram reaction
o Gram-positive cocci
o Cell arrangement
o Pairs and clusters
o Presence/Absence of PMNs
o Numerous polymorphonuclear
cells (PMNs)
Laboratory Diagnosis:
Cultural Characteristics-
Staphylococcus
aureus
Colony morphology
Smooth, butyrous,
white to yellow,
creamy
Grow well @ 18-24
hours
S. aureus may
produce hemolysis
on blood agar

S. aureus
Mannitol salt agar

• A useful selective & differential medium for recovering S.aureus

from faecal specimens when investigating staphylococcal food
poisoning.
• Can also be used to screen nasal carries.
• S.aureus:
» ferments mannitol
» Able to grow on agar containing 70-100 g/l NaCl (salt
tolerant).
» Mannitol salt agar containing 75 g/l NaCl is recommended,
particularly for isolating MRSA strains.
The high salt concentration selects for all the members of the genus
Staphylococcus. MSA is differential because the sugar mannitol is
fermented by S. aureus to acid. The pH of the medium changes which
produces a color change from red to yellow.
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DNAse Test

 PURPOSE: To identify S.aureus which produces

deoxyribonuclease (DNAse) enzyme.
 The test is particularly useful when plasma is not
available (to perform coagulase test) or results of
coagulase are difficult to interpret.

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 PRINCIPLE:
 Deoxyribonuclease hydrolyzes DNA.
 The test organism is cultured in a medium contains DNA.
 After overnight incubation, the colonies are tested for DNAse
production by flooding the plate with a weak hydrochloric acid
solution.
 The acid precipitates unhydrolyzed DNA.
 DNAse-producing colonies are therefore surrounded by clear areas
due to DNA hydrolysis.
A Positive DNase Test A negative DNase Test

Note there is breakdown of the DNA in the agar. There is a

clear zone (arrow) around the bacterial growth where there is
no longer any DNA left in the agar to precipitate out of
solution after the HCl was added.
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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
 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
Catalase Test: Interpretation

 Presence of bubbles
Positive
Staphylococci

 Absence of bubbles
Negative
Streptococci
 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
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
 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
Novobiocin Susceptibility
Test

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

 Rarely produces disease

 Found in environment and indigenous skin flora
 Catalase +
 Coagulase =
 Produces yellow pigment
 Microdase disc differentiate between Staph & Micrococcus
Schematic Diagram for Identifying
Staphylococcal Species
 Antimicrobial Susceptibility

 For non–beta-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
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
Methicillin-Resistant
Staphylococci (Cont’d)
mecA gene
Encodes penicillin-binding proteins (PBPs)
Causes drug ineffectiveness

Gold standard
Nucleic acid probe or PCR for the mec A
gene
Vancomycin-resistant
staphylococci

 VRSA= vancomycin resistant Staphylococcus aureus

 VISA= vancomycin intermediate Saphylococcus aureus

 Detection
 Vancomycin screening media
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
 Summary Micrococcaceae
Staph. aureus Staph. Staph. Micrococcus
Epidermidis saprophyticus
Colony Opaque, Opaque, Opaque, Opaque,
Morphology smooth, raised, smooth, raised, smooth, raised, smooth, raised,
entire, white- entire, gray- entire, white, bright
golden(cream) white butyrous, yellow
glossy, white-
yellow
Hemolysis Most are beta Non-hemolytic Non-hemolytic Non-hemolytic
hemolytic
Gram GPC in GPC in GPC in GPC in pairs
morphology clusters, pairs, clusters, pairs, clusters, pairs, and tetrads
short chains or short chains or short chains or
singly singly singly
Catalase Pos Pos Pos Pos
Glucose Fermenter Fermenter Fermenter Oxidizer
fermentation
Modified Neg Neg Neg Pos
Oxidase
Bacitracin Resistant Resistant Resistant Sensitive
susceptibility
(Taxo A
0.04U)
Coagulase Pos Neg Neg N/A
Production
(tube)
Clumping Pos Neg Neg Neg
factor (slide or
latex
Coagulase test)
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?ID=2037
 http://brawlinthefamily.keenspot.com/gallery/2009-10-18-breaktime/
 http://ericaandkevin.pbworks.com/w/page/5827086/Gram-Stain-and-Other-Tests
 http://faculty.matcmadison.edu/mljensen/111CourseDocs/111Review/Unit2Reviews/micr
ococcaceae_answers.htm
 http://jeeves.mmg.uci.edu/immunology/Assays/LatexAgglut.htm
 Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic
Microbiology (4th ed.). Maryland Heights, MO: Saunders.