Staphylococcus and

Micrococcus
Dr. John R. Warren
Department of Pathology
Northwestern University
Feinberg School of Medicine
June 2007
 Taxonomy of Staphylococcus
and Micrococcus
• Members of the genera Staphylococcus and
Micrococcus characterized as catalase-positive
gram-positive cocci in the family Micrococcaceae
• DNA taxonomy indicates Staphylococcus and
Micrococcus distantly related
• Staphylococcus closely related to streptococci,
enterococci, and lactobacilli
• Micrococcus related to Arthrobacter
 Pathogenic Species

• Staphylococcus aureus
• Staphylococcus epidermidis1
• Staphylococcus haemolyticus1
• Staphylococcus lugdunensis1
• Staphylococcus saprophyticus1
• Staphylococcus schleiferi1
1Variable. Other coagulase-negative

species are rarely associated with disease.

 Pathogenicity of Micrococcus
• Generally harmless saprophytes that
inhabit or contaminate the skin
• Can be opportunistic in the immuno-
compromised host (brain abscess,
meningitis, pneumonia, endocarditis,
cerebrospinal fluid shunt infection,
continuous ambulatory peritoneal
dialysis peritonitis)
 Characteristics of
Staphylococcus
• Gram-positive cocci 0.5-1.5 m in diameter
occurring singly, in pairs or short chains, and
as irregular grape-like clusters (staphyle=a
bunch of grapes)
• Catalase positive
• Growth on sheep blood and chocolate agar but
not MacConkey or eosin methleye blue agar
• No growth requirement for CO 2
 Colony Characteristics on Sheep
Blood Agar of Staphylococcus1
• Staphylococcus aureus: large (6-8 mm)
smooth, slightly raised (low convex),
cream to yellow to orange colonies with
(usually) -hemolysis
• Staphylococcus epidermidis: smaller (2.5-6
mm) white, non-pigmented colonies
without hemolysis
13 days of incubation
 Mannitol Salt Agar: Selective-
Differential Agar for Staphylococcus
aureus
• Selective and differential for recovery in culture of
Staphylococcus aureus with specimens contaminated by
commensal bacteria (nasal and rectal swabs, stool)
• Halophilic growth of Staphylococcus (7.5% NaCl)
(selective)
• Mannitol fermentation by S. aureus with acid formed
(yellow colonies due to color change of pH indicator
phenol red) (differential)
• Utility for infection control surveillance in hospital
outbreaks of staphylococcal infection
Characteristics of Micrococcus
• Gram-positive cocci 1-1.8 m in
diameter forming pairs, tetrads
(predominantly), or irregular
clusters
• Catalase positive
Characteristics of Micrococcus
• Growth on sheep blood or chocolate
agar but not MacConkey or eosin
methylene blue
• Small, convex, non-hemolytic,
variably pigmented (white, tan,
orange, yellow pink) colonies
 Staphylococcus: Natural
Habitats
• Staphylococcus aureus colonizes mucous
membranes of the anterior nares and
nasopharynx, and perineal skin
• Staphylococcus epidermidis widely
distributed on the skin
• Staphylococcus haemolyticus colonizes skin
especially areas with numerous apocrine
glands (axillae)
 Staphylococcus: Natural
Habitats
• Staphylococcus lugdunensis normal flora of
skin but in fewer numbers than
Staphylococcus epidermidis
• Staphylococcus saprophyticus colonizes the
skin, and mucous membranes of the
rectum, endocervix, and lower urinary tract
• Staphylococcus schleiferi commonly live on
carnivora
Micrococcus: Natural Habitats
• Micrococcus transient flora on exposed
skin of face, arms, hands, and legs
 Staphylococcus: Modes of
Infection
• Person-to-person transmission
by unwashed hands or fomites
• Spread of endogenous strains to
sterile sites due to medical or
traumatic disruption of skin
(wound infection)
 Staphylococcus: Modes of
Infection
• Implantation of medical devices
(cerebrospinal fluid shunts, prostheses,
peripheral and central intravascular
catheters)
• Staphylococcus saprophyticus:
introduction of endogenous strains into
sterile regions of the urinary tract
 Staphylococcus: Types of
Infectious Disease
• Staphylococcus aureus most virulent
species (obligate pathogen), commonly
causing skin infection (folliculitis,
furuncles, carbuncles), wound infection,
nosocomial pneumonia, endocarditis, and
disseminated infection (bacteremia,
osteomyelitis, myocarditis, cerebritis, and
pyelonephritis)
 Staphylococcus: Types of
Infectious Disease
• Strains of S. aureus producing toxic
shock syndrome toxin 1 (TSST-1) cause
fever, hypotension, and a blanching
rash with vomiting, diarrhea, and renal
failure (menstruating women and
highly absorbent tampons, non-
menstruating women, men)
 Staphylococcus: Types of
Infectious Disease
• Strains of S. aureus producing exfoliatins cause
excessive sloughing of the epidermidis with
fever and hypotension (scalded skin syndrome
in newborns)
• Strains of S. aureus producing enterotoxins
cause vomiting and diarrhea a few hours after
ingesting contaminated food occasionally with
hypovolemic shock due to dehydration (acute
food poisoning)
 Staphylococcus: Types of
Infectious Disease
• Strains of Staphylococcus aureus with the mecA
gene produce a low-affinity penicillin binding
protein (PBP2a, or PBP2') that imbues
methicillin resistance (MRSA) resulting in
treatment failure with oxacillin or nafcillin
• Screening Mueller-Hinton agar containing
6µg/ml oxacillin and 4% NaCl
• Direct detection by latex agglutination test for
PBP2a or polymerase chain reaction for the
mecA gene
 Staphylococcus: Types of
Infectious Disease
• Ability of the coagulase-negative
staphylococci to form a biofilm on the
surface of indwelling or implanted
foreign bodies makes them frequent
agents of intravascular infections
 Staphylococcus: Types of
Infectious Disease
• S. epidermidis causes nosocomial
bacteremia associated with intravascular
catheters, endocarditis involving
prosthetic valves, and infections of
prosthetic joints, CSF shunts, and
vascular grafts
• S. epidermidis associated with bacteremia
in neonatal intensive care units
 Staphylococcus: Types of
Infectious Disease
• S. haemolyticus causes native valve
endocarditis, bacteremia, peritonitis, and
urinary tract, wound, bone, and joint
infections
• S. lugdunensis implicated in endocarditis,
urinary tract infection, endophthalmitis, septic
arthritis, and vascular catheter infections,
associated with aggressive infections having a
high mortality
 Staphylococcus: Types of
Infectious Disease
• S. saprophyticus produces urinary tract
infection in sexually active young women
• S. schleiferi implicated in endocarditis,
septicemia, joint infections, osteomyelitis,
and wound infections
 The Coagulase Test
• Utilized with aerobic and facultative catalase-positive
gram-positive cocci
• Slide coagulase test: clumping factor with thrombin-
like activity in the cell wall of Staphylococcus aureus
reacts directly with rabbit plasma1 fibrinogen causing
fibrin formation and agglutination of bacteria by fibrin
nets (drop of rabbit plasma placed on a glass slide,
colony material emulsified in drop of plasma, visually
observed for agglutination of organisms)
1Contains EDTA
 The Coagulase Test
• Latex agglutination test1: particles coated with
fibrinogen (reacts with clumping factor),
immunoglobulin G (reacts with cell wall protein A of S.
aureus), and monoclonal antibodies against S. aureus
types 5 and 8 capsular polysaccharide (react with
encapsulated strains of S. aureus which are
predominantly methicillin resistant)
• Rapid clumping of latex particles occurs when mixed
with colony material of S. aureus from sheep blood
agar
1Variant: passive hemagglutination test in which sheep
red blood cells are sensitized with fibrinogen to detect
clumping factor
 The Coagulase Test
Tube coagulase test: extracellular (free)
coagulase complexes with rabbit plasma
coagulase reacting factor (CRF) which
generates thrombin-like activity and
coagulation (clotting) of plasma
 Staphylococcus and
Micrococcus: Identification
• Staphylococcus aureus positive for bound and
free coagulase1
• Coagulase-negative Staphylococcus
furazolidone-susceptible (100 g furazolidone
disk) and bacitracin-resistant (0.04 unit
bacitracin disk)
1Any strain negative for bound coagulase should

be confirmed with a tube coagulase test

 Staphylococcus and
Micrococcus: Identification
• Micrococcus coagulase-negative, furazolidone-
resistant, and bacitracin-susceptible
• Coagulase-negative Staphylococcus negative
for cytochrome oxidase activity measured
using the reduced substrate
tetramethylparaphenylenediamine in the
presence of dimethylsulfoxide, and
Micrococcus cytochrome oxidase positive
 Coagulase-Negative
Staphylococcus: Identification
• Polymyxin B resistance (<10 mm 300-U disk)
• Novobiocin resistance (<16mm 5-µg disk)
• Pyrrolidonyl-β-naphthylamide
• Ornithine
• Urea
• Nitrate
• Maltose, sucrose, and mannose
+ >90%, – >90%, d = 11-89% +
 Coagulase-Negative
Staphylococcus: Identification
PB NB PYR ORN UR NO3
S. epi + – – d + +
S. haem – – + – – +
S. lug1 d – + +(8h) d +
S. sapro – + – – + –
S. schlei2 – – + – – +
1Delayed + for clumping factor

2Subspecies schleiferi clumping factor +

 Coagulase-Negative
Staphylococcus: Identification
MAL SUC MANN
S. epi + + +
S. haem + + –
S. lug + + +
S. sapro + + –
S. schlei2 – – +
 Coagulase-Negative Staphylococcus:
Identification at Northwestern
Memorial Hospital

• >90% confidence level by automated Vitek 2

identification for S. epi, S. haem, S. sapro, S.
lug-Report the species identification
• <90% confidence level by Vitek 2 for S. epi, S.
haem, S. sapro, S. lug-Manual Identification
Required
• Any confidence level by Vitek 2 for S. schlei-
Manual Identification Required
 Staphylococcus aureus: strains
with weak coagulase reaction
• Colonies 6-8 mm in diameter on sheep
blood agar (24-48 h at 35o in air), white to
cream opaque (occasionally yellow to
golden), β-hemolytic
• DNase production
• S. schleiferi also + for clumping factor
and DNase, but negative for polymyxin B
resistance (Staphylococcus aureus +)
 Recommended Reading
Winn, W., Jr., Allen, S., Janda, W., Koneman,
E., Procop, G., Schreckenberger, P., Woods,
G.
Koneman’s Color Atlas and Textbook of
Diagnostic Microbiology, Sixth Edition,
Lippincott Williams & Wilkins, 2006:
• Chapter 12. Gram-Positive Cocci. Part I:
Staphylococci and Related Gram-Positive Cocci
 Recommended Reading
Murray, P., Baron, E., Jorgensen, J., Landry,
M., Pfaller, M.
Manual of Clinical Microbiology, 9th
Edition, ASM Press, 2007:
• Bannerman, T.L., and Peacock, S.J.
Chapter 28. Staphylococcus, Micrococcus,
and Other Catalase-Postive Cocci