MEDT 14 | CLIN. BACTERIOLOGY (LEC) LESSON 7.

FAMILY MICROCOCCACEAE
Jonnel P. Andaya || 3rd Year (GRAM-POSITIVE COCCI)
Transcribed by: De los Reyes, JD | Ybanez, TM

● Staphylococcus can be invasive because some strains are

able to produce Phanton-valentine-leukocidin toxin:
OUTLINE:
destroys WBC
I. INTRODUCTION ● Infections associated to Staphylococcus aureus
II. GENERAL CHARACTERISTICS OF o Bacteremia – presence of bacteria in the blood,
MICROCOCCACEAE may induce sepsis, hypotension and shock
III. CLINICAL MANIFESTATIONS o Bacterial meningitis – infection in the meningitis
IV. VIRULENCE FACTORS OF S. AUREUS o Endocarditis – especially on left-sided vulvar heart
A. ADDITIONAL VIRULENCE FACTORS OF S. disease
o Intracranial phlebitis – inflammation of the veins in
AUREUS
the brain
V. OTHER STAPHYLOCOCCUS SPECIES o Osteomyelitis and septic arthritis – joints, common
VI. LABORATORY DIAGNOSIS among pre-pubertal and children
A. BIOCHEMICAL TEST o Mastitis – inflammation of the breast tissue
B. COAGULATE TEST
C. CATALASE TEST VIRULENCE FACTORS OF S. AUREUS
D. GLUCOSE INHIBITION TEST ● Capsule: Anti-phagocytic
E. DNAse TEST ● Peptidoglycan layer: May cause platelet aggregation
(endotoxin-like activity). Is anti-phagocytic
VII. OTHER AVAILABLE DIAGNOSTIC TEST
● Exotoxins TSST-I/Entorotoxin F: responsible for toxic
VIII. ANTIMICROBIAL THERAPY shock syndrome
IX. PREVENTION ● Exfoliatin or epidermolysin toxin A and B: Cause skin
scalded syndrome
INTRODUCTION ● Enterotoxin A – E: Causes food poisoning (according to
● 4 Genera of Family Micrococcaceae book of Henry’s)
o Stomatococcus (isolated in animals) ● Phanton-valentine- Leukocidin: responsible for
o Planococcus (isolated in animals) necrotizing skin, and occasionally fatal pneumonia
o Staphylococcus (isolated in humans)
o Micrococcus (isolated in humans) ADDITIONAL VIRULENCE FACTORS OF S. AUREUS
● Coagulase: converts fibrinogen to fibrin clot
GENERAL CHARACTERISTICS OF MICROCOCCACEAE ● Lipase: causes boils and cellulitis (skin appears red and
● STAPHYLOCOCCUS swollen)
o Facultative anaerobes gram-positive bacteria ● DNase: spreading factor of bacteria
o Has a grape-like morphology ● Hyaluronidase: spreading factor of bacteria
o Is subdivided into two groups ● Staphylokinase/Fibrinolysis/Leukocidin: spreading
⮚ Coagulase – negative factor (degrades clot produced by coagulation)
● Phanton-valentine-leukocidin: lyses WBC
⮚ Coagulase – positive
● Enteroxin A-D: causes food poisoning
o Coagulase – negative species are most prominently
● Enteroxin B: causes Enterocolitis
associated with infection in humans
➢ SHEL which corresponds to: ● Enteroxin F: causes Toxic Shock Syndrome
▪ S. schleiferi
OTHER STAPHYLOCOCCUS SPECIES
▪ S. haemolyticus
▪ S. epidermidis ● S. epidermidis
▪ S. lugdunensis o are opportunistic pathogen and is prevalence as
o Transmission: person to person nosocomial pathogen more related to procedures
o Colonizers of various skin and mucosal surfaces (can and practices (coagulase negative S. bacteria)
serve as first line of defense) o associated to infections resulted from surgeries
o is most commonly encountered and more virulent
CLINICAL MANIFESTATIONS than the next three, but less than S. aureus
● S. aureus is the most virulent species of staphylococci ● S. lugdunensis
o most common in animals, but can also be seen as
● Staphylococci may cause infections in skin, wound, deep
tissue infections commonly caused by S. aureus a clinical specimen from human infections
o usually involve implantation of medical devices
● Staphylococcal infections include: folliculitis, boils,
o can produce biofilm/slimy layer which can inhibit
furuncles, carbuncles, Toxic shock syndrome, skin scalded
phagocytosis (coagulase negative, however in a
syndrome and impetigo
slide test it can be coagulase positive)
● Folliculitis: localized skin infections/inflammation of hair
● S. haemolyticus
follicles
o are beta-hemolytic
● Boils/Furuncles: infection of hair follicles or in oil glands
o S. epidermidis and micrococcus: non-hemolytic
located in a much deeper tissue
o can form biofilm
● Carbuncles: red, swollen, painful and pus-filled boils
● S. saprophyticus
connected to each other under the skin.
o is most frequently associated with community
● Toxic shock syndrome toxin: systemic effect including fever,
acquired urinary tract infections in the young
desquamation (palms and soles) and hypotension
sexually active females but is not commonly with
potentially leading to shock and death.
hospital acquired infections
● Impetigo: involves the epidermis, is typified by the
production of vesicles that rupture and crust over; looks like
a honeycomb
● Skin scalded syndrome: usually afflicts to neonates due to
exfoliatin toxins (epidermolysin A & B)
PAGE 2 MEDT 14 CLINICAL BACTERIOLOGY (LECTURE) | FAMILY MICROCOCCACEAE (GRAM-POSITIVE COCCI)

LABORATORY DIAGNOSIS
● Specimen transport and collection: No special consideration
is required for specimen collection and transport of the
organisms.
● No special considerations for processing
● Direct microscopy: All micrococcaceae produce spherical
gram-positive cells, this family of bacteria tend to divide both
longitudinal and horizontal plane, forming pairs, tetrads and
ultimately, irregular clusters.
● Gram staining observation under microscope
o Staphyloccocus: Gram positive in clusters (grape
like)
o Micrococcus: Gram positive in tetrads (M. luteus),
or in sarcinae (M. sarcinae)

NOTE: It is still essential to perform biochemical test

because gram staining is only the initial step in the
identification of bacteria. ● Colonial appearance: distinguishing characteristics (e.g.,
hemolysis) of each genus and various staphylococcal at 5%
● What is the cause of gram Variability? sheep blood. Growth on chocolate agar is similar. S. aureus
o Gram Variability happens when one is unable to yields colonies surrounded by a yellow halo on mannitol salt
identify and distinguish bacteria from one another agar. However, other Staphylococci (particularly S.
after gram staining. Such case usually occurs saprophyticus) may also ferment mannitol and thus
when the sample is taken from a patient who is resemble S. aureus on this medium.
already taking antibiotics. This supports the idea ● Loeffler’s Serum Slant: Enhances pigmentation of
that specimen collection should be done during the staphylococcus (differential medium)
acute phase of infection, and before antibiotics are 1. S. aureus- Golden Yellow
administered. 2. S. citrus- Lemon yellow
o This may also happen on old cultures (48-72 3. S. albus/ epidermidis – Porcelain white
hours). Keep in mind that one should only utilize ● Chapman’s agar/ Mannitol salt agar: Selective and
young cultures instead of those old ones. differential; Inhibitor: 7.5 % NaCl; phenol red as the
● Gram stains should be performed on young cultures, indicator: S aureus is yellow while S. epidermidis and S.
because very old cells may lose their ability to retain crystal saprophyticus is red color due to non-mannitol fermentation
violet and may appear gram variable or gram positive. ● Visible growth on 5% sheep blood and chocolate agars
● Other genera that may resemble Staphylococcus: incubated at 35 degrees Celsius in Carbon dioxide (CO2) or
A. Kocuria ambient air usually occurs within 24 hours of incubations.
B. Arthrobacter ● Mannitol salts agar and other selective media may require
C. Dermococcus incubation at least 48 to 72 hours before growth is detected.
D. Kytococcus ● Staphylococci grow well on any peptone-containing nutrient
E. Nesterenkonia medium under aerobic and anaerobic conditions and may
● Cultivation: produce hemolysis of various species of animal blood cells.
1. Media of choice: Micrococcaceae will grow on 5%
sheep blood and chocolate agars (CAP & BAP) but
not on Mac Conkey agar.
o Advantage: S. aureus can easily
grow on these agars (24 hrs)
o Disadvantage: prone to
contamination
2. Selective Media: Mannitol salt agar/Chapman’s
agar, which contains high concentration of salt (7.5
- 10 %) the sugar mannitol and phenol red as the
pH indicator. On this medium, organisms such as
S. aureus that can grow in the presence of salt and
ferment mannitol produce colonies surrounded by
yellow halo.
o Disadvantage: bacteria take 48-72
hours to grow

NOTE: Halophilic – those bacteria that can grow in

environments with high concentration of NaCl.

To answer: Which ones are mannitol and non-mannitol

fermenter?

S. aureus – mannitol fermenter (yellow)

S. epidermidis, S. saprophyticus – non mannitol fermenter
(no change in color)
* In some rare cases, S. saprophyticus can ferment mannitol
which led them to appear yellow. Hence, it is important to
confirm the identity of the bacteria using biochemical test.
Retrieved from: Baileys & scott’s diagnostic microbiology. 12th edition, by Forbes,
B.A., Sahm, D.F., & Weissfeld, A.S., (2007).
PAGE 2 MEDT 14 CLINICAL BACTERIOLOGY (LECTURE) | FAMILY MICROCOCCACEAE (GRAM-POSITIVE COCCI)

BIOCHEMICAL TEST ● Both for bacitracin and for furazolidone resistance disk are
used. A 0.04 U bacitracin impregnated disk, 100 uq
furazolidone impregnated disk are placed on the surface of
NOTE:
a 5% sheep blood agar plate that has been previously
streaked in three directions with cotton tipped swab that has
Always choose the one with most dominant colonies for
been dipped in a bacterial suspension. Prepare to match the
identification and testing. Perform gram stain (for it might be
turbidity of the 0.5 Mc. Farland standard (i.e the same as is
yeast instead of bacterial colonies).
used in preparing inocula for disk diffusion susceptibility test.
Perform subculture if there are too many identified bacteria
Staphylococcus epidermis screening plate showing
present in the mother plate. Subculture is done to create a
resistance to bacitracin (taxo A disk) and susceptible to
pure culture (only one bacteria morphotype) ready for
furazolidone.
biochemical test.

How to differentiate Staphylococcus from Micrococcus?

Staphylococcus Micrococcus
Aerobic With growth With growth
Anaerobic With growth No growth
Bacitracin Resistant Susceptible
Lysostaphin and Susceptible Resistant
Furazolidone
Glucose Utilization Fermenter Oxidizer
Microdase/Modified Negative Positive (Dark
Oxidase blue)

Note:
Retrieved from: Baileys & scott’s diagnostic microbiology. 12th edition,
Micrococcus – obligate aerobic by Forbes, B.A., Sahm, D.F., & Weissfeld, A.S., (2007).

Fermenter – With or without oxygen, staph can utilize

glucose / carbohydrates. COAGULATE TEST
● The enzyme coagulase produce by S. aureus binds plasma
Oxidizer – There is a need for oxygen to utilize glucose. fibrinogen and activates a cascade of reactions that cause
plasma clot.
For Microdase/Modified oxidase test, put bacteria on a filter
paper and pour drops of Microdase reagent. After 2 minutes, 2 types of Coagulase Test:
if it turns blue, then the sample is of Micrococcus spp. 1. Bound coagulase test (slide method): Detection
of cell bound coagulase is accomplished using
Lysostaphin – 200 uq rapid slide test (i.e., the slide coagulase test) in
which a positive test is indicated when the
organisms agglutinate on a glass slide mixed with
plasma. NOTE: Never use citrated plasma.
● Once organisms have been characterized as a gram- 2. Free coagulase test (tube method): colonies put
positive catalase-positive coccoid bacterium, complete in tube with rabbit plasma incubated at 35 – 37
identification may involve a series of test: degrees and observe coagulation after 4 hours and
1. Atmospheric requirements continue to 24 hours if no coagulation at 4 hours.
2. Resistance to 0.04 U bacitracin (Taxo A disk) (Confirmatory method)
3. Furazolidone
4. Cytochrome C as determined by the microdase
(Modified Oxidase)

Differentiation among Gram Positive, Catalase – Positive

Cocci

Retrieved from: Baileys & scott’s diagnostic microbiology. 12th edition,

by Forbes, B.A., Sahm, D.F., & Weissfeld, A.S., (2007).

● Microdase disk are available commercially. A visible amount

of growth from an 18 -24 hour-old culture is smeared on the
Retrieved from: Baileys & scott’s diagnostic microbiology. 12th edition,
disk; Micrococcus spp. turn blue within 2 minutes. by Forbes, B.A., Sahm, D.F., & Weissfeld, A.S., (2007).
PAGE 2 MEDT 14 CLINICAL BACTERIOLOGY (LECTURE) | FAMILY MICROCOCCACEAE (GRAM-POSITIVE COCCI)

VP PYRase
● Coagulase test is use to separate coagulase positive
staphylococcus from coagulase negative staphylococcus. S. aureus + -
● Isolates suspected of being S. aureus but failing to produce
bound coagulase must be tested for production of S. schleiferi + +
extracellular coagulase because S. lugdunensis, S. S. hyicus - -
schleiferi may give a positive slide coagulase test. This test,
referred to as the tube coagulase test, is performed by S. intermedius - +
inoculating a tube containing plasma and incubating at 35 *Shown on ppt presentation and is similar to the previous
degrees Celsius. Production of enzyme results in a clot photo.
formation within 1 to 4 hours of inoculation.
● Some strains produce fibrinolysis dissolve the clot after 4
hours of incubation at 35 degrees and may appear to be CATALASE TEST
negative if not read at 4 hours. ● Staphylococci are catalase positive spherical cocci that
● S. intermedius is an important agent of dog bite wound often appear in grape-like clusters in stained smears.
infections and may be misidentified as S. aureus if only ● Catalase Test:
coagulase testing is performed. Microbiologists may want to o Differentiate Staphylococcus from Streptococcus
consider performing the additional test in cases in which o Colony added in reagent 3% H2O2 (hydrogen
coagulase positive staphylococci are isolated from dog bite peroxide)
wound infection. o Positive result is bubble formation
● It is particularly important to differentiate S. lugdunensis o Do not get colony from BAP due to
from other coagulase-negative staphylococci from sterile pseudoperoxidase activity of Hgb which may
sites because there are different interpretative criteria for cause a false positive result.; Avoid getting the
oxacillin for organisms. S. lugdunensis is 2 HR PYR positive BAP agar medium only the colonies must be
and ornithine decarboxylase positive. applied to the reagents.
● S. aureus produce coagulase an enzyme that binds plasma
fibrinogen causing the organisms to agglutinate or plasma
to clot.
● S. aureus is 95% identified by the slide coagulase test and
100% of all isolates are identified by tube coagulase test,
which detects free coagulase – one being released or
excreted by coagulase positive Staphylococcus.
● S. lugdunensis and S. schleiferi are primary pathogen of
animals and rarely encounter to humans.
● Tube coagulase test incubated at 35 degrees for 4 HRS
(initial reading), if no clot has formed the tube is reincubated
at room temperature and reexamined after a total of 24 Retrieved from: Baileys & scott’s diagnostic microbiology. 12th edition,
hours of incubation. NOTE: 0.5 commercially-prepared rabid by Forbes, B.A., Sahm, D.F., & Weissfeld, A.S., (2007).
plasma mixed with water is used. 0.05 rabid plasma is added
to the tube.
● False positive coagulase test: Presence of citrate utilizing
bacteria that cause the plasma to clot. Citrate is an GLUCOSE INHIBITION TEST
anticoagulant. ● Oxidation – fermentation test (OF test): medium is OF
● Coagulase negative staphylococcus medium
o S. epidermidis: novobiocin susceptible ● Carbohydrates component: glucose
o S. saprophyticus: novobiocin resistant ● pH indicator: Bromthymol blue
● To differentiate Staphylococcus from other coagulase ● Color reactions: Yellow (Acid production), Blue (alkaline)
positive/variable staphylococcus see image below: NOTE: Add mineral oil (black on figure) on one tube then
incubate to identify if a sample is Staph or Micrococcus.

Retrieved from: Baileys & scott’s diagnostic microbiology. 12th edition,

by Forbes, B.A., Sahm, D.F., & Weissfeld, A.S., (2007).
PAGE 2 MEDT 14 CLINICAL BACTERIOLOGY (LECTURE) | FAMILY MICROCOCCACEAE (GRAM-POSITIVE COCCI)

DNAse TEST (DNA Hydrolysis Test)

● Is a supplementary test to further confirm if the sample is S.
aureus
● Medium: DNA agar
● Dye: methyl green

*If there is clearing, then it is DNase positive.

Retrieved from: Baileys & scott’s diagnostic microbiology. 12th edition,

by Forbes, B.A., Sahm, D.F., & Weissfeld, A.S., (2007).

OTHER AVAILABLE DIAGNOSTIC TEST

● Latex agglutination procedures that detect clumping factor
and protein A
● Passive hemagglutination test that detect clumping factor
● Latex agglutination and Passive hemagglutination are
no longer in vogue because of failure to detect MRSA
(Methicillin Resistant Staphylococcus aureus)
● Antibodies to techoic acid a major cell wall component of
gram-positive bacteria are usually produced in long standing
or deep-seated staphylococcus infection such as
osteomyelitis. This procedure is usually performed in
reference laboratory, however the clinical utility of
performing this assay is at best uncertain.

ANTIMICROBIAL THERAPY
● Staphylococci Therapy
o Several agents from major class of antimicrobials
including aminoglycosides, beta-lactams,
quinolones and vancomycin.
o For many isolates a penicillinase-resistant
penicillin (e.g., nafcillin, oxacillin, methicillin-if
bacteria are resistant to beta-lactams) is used.
o Vancomycin is used when isolates resistant to
these penicillin derivatives are encountered.

PREVENTION
● No approved anti-staphylococcal vaccines.
● Intranasal carriers of an epidemic strain of S. aureus are
treated with mupirocil and some with rifampin.
● Some physicians advocate the use of antibacterial
substances such as gential violet, acriflavine,
chlorhexidine or bacitracin to the umbilical cord stump to
prevent staphylococcal disease in hospital nurseries.
● During epidemics it is recommended that all full-term infants
be bathed with 3% hexachlorophene as soon after birth as
possible and daily thereafter until discharge.

References:
• Forbes, B.A., Sahm, D.F., & Weissfeld,
A.S.(2007).Baileys & Scott’s Diagnostic
Microbiology.12 th ed.USA: Elsevier.

• McPherson, & R.A.,Pincus, M.R.,(2011). Henry’s

Clinical Diagnosis and Management by Laboratory
Methods. 22nd ed. USA :Elsevier.