CLINICAL MICROBIOLOGY

Chapter 14
STAPHYLOCOCCI
TAXONOMY
Coag
POS

Staphylococcace Staphylococc
ae us
Coag
NEG

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GENUS
STAPHYLOCOCCUS
Coagulase
Coagulase postive Negative

S. aureus S.epidermidis
S. saprophyticus
S. haemolyticus

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STAPHYLOCOCCUS: GRAM
REACTION & MORPHOLOGY
Gram positive,
spherical cells (0.5-1.5
µm) in singles, pairs
and clusters

“Bunches of grapes”

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STAPHYLOCOCCUS: GENERAL
CHARACTERISTICS
➔ Nonmotile ➔ Glucose
➔ Non spore- fermenters
forming ➔ Primarily aerobic
➔ Nonencapsulated ➔ Some facultative
➔ Catalase anaerobes
producing
➔ Oxidase negative
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STAPH: GENERAL
CHARACTERISTICS (cont’d)
➔ Bacitracin resistant
➔ Grow on peptone
agar
➔ Inhibited by bile salt
media

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STAPH: GENERAL
CHARACTERISTICS (cont’d)
➔ Colony morphology:
◆ Medium sized (4-
8mm)
◆ cream/white/golde
n yellow
◆ “buttery”
➔ Can be β-hemolytic

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PRIMARY PATHOGEN:
Staphylococcus aureus
➔ Primary reservoir is the
human nares
➔ Can also colonize the
axillae, vagina, pharynx,
and other skin surfaces
➔ Superficial to systemic
infections

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Staphylococcus aureus
➔ Mode of transmission
◆ Traumatic introduction
◆ Direct contact
◆ Inanimate objects
➔ Predisposing conditions
◆ Indwelling devices
◆ Immune response defects

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Staphylococcus aureus
➔ Suppurative infection
◆ Bypass initial host barriers
◆ Activate host’s inflammatory
response
◆ Resist/kill inflammatory cells
◆ Copious amount of pus produced
◆ Pus consists of damaged/dead
cells, extravascular fluid and
organisms
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VIRULENCE
FACTORS:Staphylococcus aureus
➔ Enterotoxins
◆ Can cause vomiting and diarrhea
◆ Heat stable @100°C for 30
minutes
◆ Food poisoning

➔ Toxic Shock Syndrome Toxin-1 (TSST)

◆ Causes nearly all menstruation-
associated TSS
◆ It is a superantigen stimulating T-
cell proliferation

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VIRULENCE
FACTORS:Staphylococcus aureus
➔ Exfoliatin toxin
◆ Staphylococcal SSS or Ritter
disease

➔ Cytolytic Toxins
◆ 4 hemolysins
◆ 1 leukocidin: Panton-Valentine
leukocidin (PVL)

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VIRULENCE
FACTORS:Staphylococcus aureus
➔ Extracellular Enzymes
◆ Hyaluronidase
◆ Lipase
◆ Staphylocoagulase
◆ Protein A
● Cell wall component
● Binds to Fc of IgG
● Blocks phagocytosis

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SKIN/WOUND INFECTIONS:
Staphylococcus aureus
➔ Folliculitis
➔ Furuncles (boils)
➔ Carbuncles
➔ Impetigo
➔ Scalded Skin
syndrome/Ritter disease

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MULTI-SYSTEM DISEASE:
Staphylococcus aureus
➔ Toxic Shock Syndrome
◆ Rare; potentially fatal
◆ Sudden onset of high
fever, vomiting, diarrhea,
muscle aches
◆ Can progress to
hypotension and shock
◆ Can affect men, women
and children
◆ TSST-1 toxin is systemic

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FOOD POISONING:
Staphylococcus aureus
➔ Staphylococcal food poisoning
◆ Source: infected food handler
◆ Caused by enterotoxin
◆ Most commonly affected foods
are those with mayonnaise or
eggs.
◆ Onset of symptoms in 2-8 hours
◆ Resolution in 24-48 hours

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COAGULASE NEGATIVE STAPH
➔ Often found as normal flora
➔ Presence can indicate contamination
➔ Predisposing factors to infection:
◆ Catheterization
◆ Medical device implantation
◆ Immunosuppressive therapy
➔ Abbreviated CNS or CoNS

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COAGULASE NEGATIVE STAPH:
S.epidermidis
➔ Predominantly HAIs
◆ Skin flora introduced by
catheters, CSF shunts, and
heart valves
◆ Common cause of healthcare
acquired UTIs
◆ Biofilm production critical to
pathogenesis
◆ Septicemia in
immunocompromised patients

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COAGULASE NEGATIVE STAPH:
S.saprophyticus
➔ UTIs in young, sexually active
women
◆ Adheres better vs other CNS
to urogenital epis
◆ Even low numbers in urine
culture are considered
significant
◆ Rarely on other skin/mucous
membranes

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COAGULASE NEGATIVE STAPH:
Other CNS
➔ MANY other CNS species
➔ Many also found as normal flora
➔ Often opportunistic pathogens
➔ S.lugdunensis and S.haemolyticus
are other commonly isolated CNS.

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 LABORATORY DIAGNOSIS
➔ Specimen collection/handling
◆ Specimen from site of infection
◆ Avoid normal flora
contamination
◆ Prevent delay in transport
➔ Microscopic examination
◆ Gram positive cocci
◆ PMNs
◆ Genus/species ID not based
on microscopic morphology
alone

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LABORATORY DIAGNOSIS:
S.aureus

➔ Colony morphology
◆ White to yellow, creamy,
buttery
◆ Growth 18-24 hours at 37°C
◆ Possible β-hemolysis

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LABORATORY DIAGNOSIS:
S.epidermidis, S.
saprophyticus
➔ Colony morphology: S.epidermidis
◆ Small-medium sized
◆ Gray to white colonies
◆ Nonhemolytic or weak
hemolysis

➔ Colony morphology:
S.saprophyticus
◆ Medium sized
◆ White to slight yellow

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LABORATORY DIAGNOSIS:
Identification Tests
➔ Catalase
◆ Differentiate Staph vs. Strep
◆ Staph: catalase POSITIVE
◆ Strep: catalase NEGATIVE
◆ Procedure:
● Smear colony on slide
● Add H2O2
● Observe for bubbles
(POS)
● CAVEAT: No blood agar!
Can lead to false POS.

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LABORATORY DIAGNOSIS:
Identification Tests
➔ Slide Coagulase
◆ Detects clumping factor
◆ Fibrinogen → fibrin
◆ S.aureus: POSITIVE
◆ CNS: NEGATIVE
◆ Procedure:
● Emulsify a colony
● Add rabbit plasma
● Observe for agglutination
(POS)

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LABORATORY DIAGNOSIS:
Identification Tests
➔ Tube Coagulase
◆ Detects staphylocoagulase/free
coagulase
◆ S.aureus: POSITIVE
◆ CNS: NEGATIVE
◆ Procedure:
● Organism + rabbit plasma at
37°C
● Observe every 30 minutes for the
presence of a clot (POS)
● If NEG after 4 hours, hold at RT
up to 24 hours.

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LABORATORY DIAGNOSIS:
Identification Tests
➔ Rapid Coagulase
◆ Detect clumping factor and Protein A
◆ Employs plasma-coated latex
◆ S.aureus: POSITIVE
◆ CNS: NEGATIVE
◆ @ACC: Staphyloslide
◆ Procedure:
● Manufacturer specific
● Agglutination is POS

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LABORATORY DIAGNOSIS:
Identification Tests
➔ Novobiocin Susceptibility
◆ Only for urine isolates
◆ Differentiate S.saprophyticus
vs. other CNS
◆ Paper disc impregnated with
5g Novobiocin antibiotic
◆ S.saprophyticus: RESISTANT
(<16 mm)
◆ Other CNS: SUSCEPTIBLE
(>16 mm)

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RELATED GENUS: Micrococcus
◆ Staphylococcus previously In
Micrococcaceae family with
Micrococcus
◆ Micrococcus are indigenous skin
flora
◆ COLONY: bright, lemon yellow
colonies
◆ Catalase POS, coagulase NEG
◆ ID via microdase disc. Smear
organism on disc, Color change
to blue/purple is POS. No color
change is NEG.

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ANTIMICROBIAL SUSCEPTIBILITY
TESTING (AST)
➔ Penicillin was drug of choice to treat S.aureus
➔ Nearly all strains now penicillin resistant
➔ Owed to beta-lactamase enzyme
➔ Methicillin: penicillinase-resistant antibiotic
➔ Now MRSA

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Methicillin Resistant
Staphylococci
➔ Methicillin Resistant S.epidermidis
(MRSE)
➔ Methicillin Resistant S.aureus (MRSA)
◆ HA-MRSA vs. CA-MRSA
◆ Infection control practices
● Barrier protection
● Contact isolation
● Handwashing compliance
◆ Oxacillin resistance due to mecA
gene
◆ mecA gene encodes altered PBP
◆ Detection: nucleic acid probes or PCR

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Vancomycin Resistant
Staphylococci
➔ Vancomycin resistant S.aureus
(VRSA)
➔ Vancomycin intermediate
S.aureus (VISA)
◆ Serious gram positive
infections: Vancomycin
◆ 1987 VRE
◆ 1996 VRSA in Japan
◆ 1997 VISA in US
◆ 2002 VRSA in US
◆ Screen with vancomycin agar
plate
◆ Requires confirmation

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Macrolide Resistance:
Staphylococci
➔ Inducible Resistance to
Clindamycin
◆ Erythromycin and Clindamycin
results usually the same
◆ If erythro is R but clinda is SS,
perform D-test
◆ D-test POSITIVE: Inducible
clindamycin resistant detected;
D-shaped zone of inhibition
between discs
◆ D-test NEGATIVE: No inducible
clindamycin resistance
detected; erythro is R, clinda is
SS
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SUMMARY:
Staphylococcac
eae
&
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?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/mi
crococcaceae_answers.htm
➔ http://jeeves.mmg.uci.edu/immunology/Assays/LatexAgglut.htm
➔ Mahon, C. R., Lehman, D. C., & Manuselis, G. (2014). Textbook of Diagnostic Microbiology
(5th ed.). Maryland Heights, MO: Saunders.

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