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Micrococcaceae
Chapter 14 Staphylococci
Family Micrococcaceae
• Includes 4 genera:
– Planococcus – free living saprophyte
– Micrococcus – free living saprophyte
– Stomatococcus – normal flora on surface of
primates and other mammals
– Staphylococcus – normal flora on surface of
primates and other mammals
• All, except Planococcus, isolated from
clinically significant sources
Infection and Disease
• Stomatococci – oral normal flora (NF),
emerging pathogen in immunocompromised
• Micrococci - pathogen when accidentally
introduced into susceptible host
• Staphylococci - long recognized as
important human pathogen, most commonly
isolated species in order of pathogenicity:
– S. aureus
– S. epidermidis
– S. saprophyticus.
Gram Stain
• Gram (+) coccus
• Lose ability to retain
Gram (+) staining
characteristic with
longer culture
• Occur singly, pairs,
tetrads (common for
Micrococci), or
clusters
• Staphylococci –
staphyle (bunch of
grapes), arrangement
due to cell division in
different planes
Laboratory Culture
• Grow on most lab media that support
growth of Gram (+) microorganisms (MO)
• Overnight culture: smooth, circular,
buttery colonies
• S. aureus - golden or yellow, many clinical
isolates creamy
• S. epidermidis - white
• M. luteus - bright yellow
Differential Media: Columbia
Blood Agar (CBA)
• S. aureus produces
complete (beta) hemolysis
• Other Staphylococcus
species produce partial
(alpha) hemolysis or no
hemolysis (gamma)
Selective Media: PEA, CNA
• Used to isolate Staph.
from specimens heavily
contaminated
• Phenyl Ethyl Alcohol
(PEA) – inhibit Gram (-)
bacteria
• Columbia CNA Agar antibiotics Colistin and
Nalidixic Acid to
inhibit Gram (-)
bacteria
Differential & Selective
Media: Mannitol Salt Agar
(MSA)
• High salt (7.5%) inhibit growth
most MO
• Staph. are halophiles and grow in
up to 10% NaCl
• Also contains mannitol and pH
indicator phenol red (pH 7 = red)
• If MO growing on MSA ferments
mannitol, the acid produced turns
phenol red = yellow
• S. aureus ferments mannitol
• S. epidermidis does not
Biochemical Identification:
Catalase Test
• Stomatococci = catalase (-)
• Micrococci and Staphylococci =
catalase (+)
Oxidative/Fermentative
(O/F ) Media
• Test done using two tubes of O/F:
– Oxidation - open to air; (+) = yellow
– Fermentation – overlay with mineral oil,
absence of O2; (+) = yellow
• Micrococci = oxidative or inert
(asaccharolytic)
• Staphylococci = oxidative and
fermentative
Biochemical Test:
Micrococci vs Staphylococci
• Modified Oxidase Test (contains DMSO allows
penetration of test reagent through thick G+
cell wall):
– Micrococci = (+)
– Staphylococci = (–)
• Lysostaphin (protease breaks glycine peptide
linkage in cell:
– Micrococci = Resistant
– Staphylococci = Sensitive
• Bacitracin – (0.04 units,
• Taxo A disc):
– Micrococci = Sensitive
– Staphylococci = Resistant
ID Staphylococcus aureus:
Coagulase Test
• In presence of coagulase, fibrinogen converted to
fibrin
• Staphylococcus aureus = coagulase (+)
• Other species of Staph. are coagulase positive, but
are rare isolates from human infections (i. e., S.
intermedius from canine bites)
• Therefore, (+) coagulase test usually identifes isolate
as S. aureus
• All other Staph species collectively called coagulase
negative Staph. (CoNS), these include S. epidermidis
and S. saprophyticus
Two Coagulase Test
• Slide test for bound coagulase
• Tube test test for free or
extracellular coagulase
Differentiation of
Staphylococcus
• Mannitol fermentation
– S. aureus and some S.
saprophyticus = (+)
– S. epidermidis = (-)
• DNAse
– S. aureus = (+)
– S. epidermidis and S.
saprophyticus = (-)
Differentiation of Coagulase
Negative Staphylococcus:
Novobiocin
• Usually for urinary tract isolates
• ID S. epidermidis from S.
saprophyticus
• Novobiocin (5 ug disk)
• S. epidermidis = Sensitive, zone
of inhibition ≥17 mm. in diameter
• S. saprophyticus = Resistant,
zone <17 mm. in diameter
Serology & Phage Test
• Antibody titer to Staph. teichoic acids
(part of the cell wall); rising titer may be
used to confirm diagnosis of Staph.
endocarditis
• Epidemiologic investigations identify
different strains of S. aureus based on
type of antigenic polysaccharide capsule
• Phage typing – S. aureus and S. epidermidis
strains placed into different groups based
on susceptibility by different
bacteriophages; used in epidemiologic
investigations
Mechanisms of Pathogenicity:
Staphylococcus aureus
• Capsule – allows MO to resist phagocytosis;
some only form capsules “in vivo”
• Teichoic acids –a fibronectin binding protein;
involved in adherence, as fibronectin on surface
of many cells
• Protein A – binds to Fc region of IgG; inhibits
phagocytosis by preventing opsonization
(antibody-enhanced phagocytosis)
S. aureus Exotoxins
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Hemolysin
Panton-Valentine Leukocidan
Enterotoxin
Exfoliative
Toxic Shock Syndrome Toxin (TSST)
Exotoxins
• Hemolysin – S. aureus is beta hemolytic:
– May produce 4 different hemolysins: ,, , 
– Except for  hemolysin, they lyse RBCs and
also leukocytes and tissue cells
–  toxin, in particular, produce extensive tissue
damage
–  hemolysin known as hot-cold lysin; hemolytic
activity enhanced when 370 C followed by 40 C
incubation
• Panton-Valentine Leukocidan:
– Acts exclusively on WBCs (PMNs and
macrophages)
– Lytic activity due to alteration of Na+ - K+
pump, leads to altered permeability and
eventual lysis of cell
Exotoxins
• Enterotoxins – 8 distinct enterotoxins responsible for
Staph. food poisoning:
– Heat stable
– Stimulate neural receptors in G.I. tract causing pain,
vomiting, diarrhea within 6 hours of ingestion
– Symptoms short lived
– Former type F (see below) now known as toxic shock
syndrome toxin (TSST-1)
• Exfoliative (Epidermolytic) toxin:
– Cleaves upper layer of the epidermis
– Condition called scalded skin syndrome (SSS)
• TSST– 1:
– Pyrogenic (fever causing) due to IL-1 induction
– Erythroderma (red skin)
– Enhanced susceptibility to endotoxin shock
• Many of the effects of enterotoxin, exfoliative toxin
and TSST-1 are due to their action as a superantigen
Action of a Superantigen
• Non-specific stimulation of T cells
• Leading to cytokine release
(“cytokine storm”)
• Causes inflammation, leads to fever,
hypotension and shock
S. aureus Enzymes
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Coagulase
Staphylokinase
Hyaluronidase
Lipase
Beta lactamase
DNAse
Slime production
Enzymes: Coagulase
• Helps to wall off MO from host immune system
• Also help by coating neutrophils with fibrin to
protect MO from phagocytosis
• Many Staph. infections are characterized by
abscess formation
• Free or extracellular coagulase combines with
serum component to produce thrombin-like
activity to cleave fibrinogen to form fibrin clot
• Bound coagulase binds to fibrinogen on cell
surfaces converting it to fibrin, producing fibrin
clots and causing agglutination of the bacterial
cells (also called clumping factor)
• High concentrations of coagulase can lead to
intravascular coagulation, particularly in the lungs
Enzymes
• Staphylokinase – dissolves fibrin clots that host
may lay down during an inflammatory reaction to
try to wall off the infection; help MO to
disseminate
• Hyaluronidase – depolymerizes hyaluronic acid,
the ground substance of tissues; help MO to
disseminate
• Lipase – hydrolyzes lipids; help MO to disseminate
• Beta lactamase (Cefinase disks) – breaks beta
lactam ring to inactivate penicillin
Enzymes
• DNAse – degrades
accumulated inflammatory
exudate DNA from leukocyte
disintegration, helping MO to
spread (DNA very viscous
making dissemination difficult)
• Slime production –
extracellular glycoconjugate
helps MO to adhere to smooth
surfaces and produced by
CNS; important in colonization
of indwelling catheters
Summary: S. aureus
Virulence Factors
Clinical Significance of
Staphylococcus
• Ubiquitous, found as NF of humans and
other animals
• CoNS strains (usually S. epidermidis) NF
of skin
• S. aureus NF of nasopharynx in 10-40% of
population; percentage higher in
hospitalized patients
• Opportunistic (S. epidermidis) or
Facultative (S. aureus) pathogens
S. aureus : Invasive Infections
• Cause localized infections in
nearly any area of body
• Local skin infections most
common
• Suppuration (pus production)
hallmark of these infections
• Folliculitis - infection of hair
follicle; if hair follicle is
eyelash, infection called a stye
• Furuncle or boil – folliculitis
spreads to involve
subcutaneous tissue
• Carbuncle – series of
interconnected furuncles
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Invasive Infections
Spreads throughout body
Bacteremia
Septicemia with lymphangitis
Osteomyelitis
Pneumonia
Meningitis
Endocarditis – acute or subacute (can
occur following tooth extraction)
• S. aureus and S. saprophyticus cause
urinary tract infection
• S. saprophyticus is second most
common cause of urinary tract
infection in sexually active young
women
S. aureus :Toxigenic Diseases
• Food poisoning due to heat stable enterotoxin; more
common in foods with mayonnaise or custard
• Toxic shock syndrome – commonly starts as vaginal
infection in menstruating women using absorbent
tampons; sudden onset of high fever, vomiting,
diarrhea, red rash, shock due to TSST-1; followed 1-2
weeks by desquamation of palms of hands and soles of
feet
• Scalded skin syndrome:
– Exfoliative toxin, initially causes red rash, followed by peeling
away of the skin in sheaths
– Usually occurs 2 times, but heals without scarring
– More common in infants and young children
Summary: Staphylococcus
Infections
Antimicrobial Treatment
• Most S. aureus (85%) now produce beta lactamase to
inactivate penicillin
• Methicillin, a beta lactamase resistant penicillin used
• Now methicillin resistant strains of S. aureus (MRSA):
– change in cell wall and penicillin binding proteins (enzymes
for peptidoglycan cross-linking)
– altered binding of antibiotics, no longer inhibit enzymes
– also resistance to cephalosporins, streptomycin,
tetracycline, sulfonamides
• For MRSA use of vancomycin:
– given I.V.,requires hospitalization
– Vancomycin resistant strains have now been reported!
• Other Staphylococcus species tend to be even more
resistant to antibiotics than S. aureus, so antimicrobial
sensitivity testing essential
Experimental Treatment:
Bacterial Interference
• For individuals with chronic
infections of S. aureus
• The individual is colonized with a S.
aureus strain of low virulence
• Idea that no superinfection will occur
if the individual is already colonized
Class Assignment
• Textbook Reading: Chapter 14
Staphylococci
• Key Terms
• Learning Assessment Questions
Case Study 1 Staphylococcus
• An 18-year-old man fell on his knee while
playing basketball. The knee was painful,
but the overlying skin was unbroken.
• The knee was swollen and remained painful
the next day, so he was taken to the local
emergency department.
• Clear fluid was aspirated from the knee,
and the physician prescribed symptomatic
treatment.
Case Study 1
• Two days later, the swelling returned, the
pain increased, and erythema developed
over the knee.
• Because the patient also felt systemically
ill and had an oral temperature of 38.8°C,
he returned to the emergency department.
• Aspiration of the knee yielded cloudy
fluid, and cultures of the fluid and blood
were positive for S. aureus.
Case Study 1 - Questions
• 1. Name two possible sources of this
organism.
• 2. Staphylococci cause a variety of
diseases, including cutaneous
infections, endocarditis, food
poisoning, SSS, and TSS. How do the
clinical symptoms of these diseases
differ from the infection in this
patient? Which of these diseases
are intoxications?
Case Study 1 - Questions
• 3. What toxins have been implicated in
staphylococcal diseases? Which
staphylococcal enzymes have been
proposed as virulence factors?
• 4. What is the antibiotic of choice for
treating staphylococcal infections? (Give
two examples.)
• Written Report:
– Short reply to each question (2-3 sentences)
– Due Jan. 11 in lecture
– No late reports or email