GRAM-POSITIVE COCCI
_________________ – demonstrates
College of Allied Medical Professions
Lyceum of the Philippines University-Batangas
FAMILY MICROCOCCACEAE
PYOGENIC (PUS FORMING COCCI)
I. STAPHYLOCOCCI
General Characteristics:
 gram (+) cocci _________________
appearance
 generally __________________________
 either aerobic / facultative anaerobic /
microaerophilic
 strongly ______________
 pus formation
Staphylococcus Micrococcus
Lysostaphin
Furazolidone
0.04 U Bacitracin
Microdase
Aerotolerance
Important species:
1. Staphylococcus aureus
2. Staphylococcus epidermidis
3. Staphylococcus saprophyticus
Staphylococcus aureus
 _______________________________
 _______________ which is soluble in ether
and alcohol and is termed as lipochrome
 normally found on the skin, mucous
membrane, mouth, nose and intestines
Cultural Characteristics:
 broth: __________________
 plated media: opaque, circular, smooth with
a butyrous consistency, oil paint
appearance,
 BAP: _____________ colonies;
__________________
Pathogenicity Tests for S. aureus:
1. Coagulase test – best single criterion for the
pathogenicity
Medium used: fresh human plasma
reconstituted citrated rabbit’s plasma
Methods of Coagulase Testing:
a.
bound coagulase or clumping factor
Procedure:
a drop of plasma + loopful of emulsified
on a slide organisms
b. _________________
Procedure:
0.5mL of plasma + inoculum 37oC
 clot formation within 4 hours
2. Growth on Tellurite Medium
S. aureus – able to reduce tellurite producing
_____________ colonies
Other staphylococci – do not reduce tellurite
but produce _________ colonies
3. Mannitol Fermentation Test
S. aureus – ferments mannitol; able to tolerate
high conc. of salt (_______ NaCl)
Medium: ________________
ph indicator: ______________
(+) result: ____________________________
4. Protein A
S. aureus – has protein A in their cell wall
Other staph – has protein B in their cell wall
5. Lysostaphin Sensitivity
A 1:10 dilution of culture is exposed to 2ug of
lysostaphin for 30 mins at 37oC.
6. Alpha-hemolysis
Serve as an index of virulence because the
more virulent the strain, the more alpha
hemolysis is produced.
Extracellular Enzymes produced by S. aureus:
1. Coagulase – brings about the clotting of
plasma; has __________ activity
2. Staphylokinase – has ___________ activity
3. Hyaluronidase – hydrolyzes the hyaluronic
acid present in the intracellular ground
substance of connective tissue, thereby
facilitating spread of infection
S. aureus – _________________
4. Lipase – hydrolyzes fats and oils on the
surface area of the body
or 5. Nuclease – a phosphodiesterase with both
endonucleolytic and exonucleolytic properties
and can cleave either DNA or RNA
Toxins:
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1. Cytolytic toxin
2. Alpha toxin (α-Hemolysin)
3. Beta toxin
4. Delta Toxin (δ-Toxin)
5. Gamma Toxin (γ-Toxin)
6. Leucocidin – attacks ____________
S. aureus – _______________
7. Enterotoxin – responsible for staphylococcal
______________
8. Exfoliative Toxin (Epidermolytic Toxin) –
responsible for _________________
9. Toxic Shock Syndrome Toxin (TSST)
Laboratory Diagnosis:
1. Perform GS to demonstrate gram (+) cocci
occurring in clusters.
2. Culture the microorganisms.
3. Perform the catalase test.
1ml of ____________ is allowed to flow over
the growing colonies.
4. Perform the confirmatory test /Coagulase test.
5. Perform the DNAse test.
6. ____________ – susceptibility of staphylococci
to symbiotic bacteriophage; used to detect the
exact strains of staphylococci causing
epidemic outbreaks
B. Staphylococcus epidermidis
 formerly _____________
 produces ________________ of colonies
on blood agar
 non-pathogenic
 low in virulence as compared to S. aureus
 __________________________
 associated with mild “stitch” abscesses
 commensal of the human skin; pathogenic
if implanted on other areas other than skin
 clinically important in _______________
after cardiac surgery
 coagulase (-)
 MFT (-)
 non-hemolytic
 does not contain Protein A
C. Staphylococcus saprophyticus
 occurs in __________________________
 common cause of _____ in young sexually
active women; 2nd to E. coli
 non-hemolytic
 does not contain Protein A
 coagulase (-)
 MFT (+)
 failure to ferment glucose anaerobically
 ____________________
 ____________________
Clinical Manifestations of Staphylococci:
_______________ – the characteristic feature of
staphylococcal infection
1. Cutaneous Infections
a. ___________– infection of the hair follicle
b. _____________ – focal suppurative lesion
that is extended into the subcutaneous
tissue
c. ___________ – similar to furuncle but has
multiple foci and extends into the deeper
layers of fibrous tissue; limited to the neck
and upper back where the skin is thick and
elastic
d. _________ – characterized by the
formation of encrusted pustules on the
superficial layers of the skin; common in
young children, often occurring around the
nose; highly contagious
e. ______________________
Three Distinct But Related Clinical Entities:
a. Generalized Exfoliative Dermatitis
(____________________, staphylococcal
toxic epidermal necrolysis) – characterized
by generalized painful erythema and
dramatic bulllous desquamation of large
areas of skin
b. Bullous impetigo – a localized form of the
syndrome in which the infection occurs at
the site of lesion
c. Staphylococcal Scarlet Fever – mild
generalized form of the scalded skin
syndrome, clinically similar to streptococcal
scarlet fever
2. Pneumonia – very important disease because
of its high mortality rate (up to 50%)
3. Osteomyelitis – the organisms localize at the
diaphysis of long bones
4. Pyoarthrosis – an infection with S. aureus that
occurs near the joint
5. Bacteremia and Endocarditis
6. Food Poisoning
7. Toxic Shock Syndrome (TSS)
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II. STREPTOCOCCI c. Gamma-hemolytic Streptococci /
General Characteristics: Nonhemolytic Streptococci / Indifferent
 gram (+) in _______ and in _________ Streptococci
 facultative anaerobic  produce no hemolysis
 requiring 10% CO2 for initial isolation
 homolactic fermenters III. Lancefield Classification
 catalase (-)  based on the presence of serologically
 NS, NM, NE except _______________ active carbohydrate known as
 pus formation ______________________
A. Group A
Cultural Characteristics:  primarily human pathogen
 _______________ requiring enriched E.g. ____________ – always β-hemolytic
media like BAP ___________ – virulence factor
 form ______________ translucent to
slightly opaque colonies Presumptive Tests to Identify Group A
 some strains exhibit _______________ β-hemolytic Streptococci:
1. Bacitracin Disc Test
Three General Classifications of Streptococci: Principle:
I. Academic/Bergey’s Classification Based on the selective inhibition of the
 based on the physiological division of growth of Group A β-hemolytic streptococcus
streptococci by a paper disc containing 0.04U of Bacitracin
a. _____________ – grow at neither 10oC nor (+) : ____________________
at 45oC
E.g. S. pyogenes 2. PYR Test
b. _________ – grow at 45oC but not at 10oC Principle:
E.g. S. mutans Based on the hydrolysis of L-pyrolidonyl B-
S. salivarius napthylamide
c. ____________ – grow at 10oC and 45oC (+) : ____________________
E.g. S. faecalis
d. ___________ – grow at 10oC but not at B. Group B
45oC ___________________
E.g. S. lactis  the only member
 important agent of bovine mastitis
II. Smith and Brown’s Classification  the organisms in female genitalia which
 based on the ______________________ can colonize neonates
a. Alpha-hemolytic Streptococci / Green  causative agent of ________________
Streptococci  rapidly hydrolyzes ______________
 produce ________ hemolysis of red  produces a protein-like factor
blood cells accompanied by a compound known as ____________
_____________________ possibly that enhances the β-hemolytic activity
due to methemoglobin of S. aureus
E.g. S. pneumoniae  normally found in the following:
Viridans streptococci 1. URT
b. Beta-hemolytic Streptococci / 2. GIT
Streptococcus hemolyticus 3. GUT
 produce a _________ hemolysis
accompanied by _________________ C. Group C
of hemolysis around the colony  primarily animal pathogen
E.g. S. pyogenes 1. ________ – causes disease in horses
S. agalactiae 2. _____________ – source of streptokinase
used in thrombolytic therapy in human
3. S. zooepidemicus
4. S. dysgalactiae
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D. Group D a. Streptolysin O (SLO)
 normally found in the _________  responsible for the β-hemolytic reaction
 only group capable of growing in on blood agar
_______________  inactivated by ___________
 irreversibly inactivated by __________
Subgroups:  toxic to red and white blood cells
1. Non-enterococcal various tissue culture cells
a. S. bovis b. Streptolysin S (SLS)
b. S. equinus c. DNAses A, B, C and D

2. Enterococcal Clinical Infection:

a. ____________________ – frequently
associated with urinary and biliary tract
infections, septicemia, endocarditis,
wound infection, and intra-abdominal
abscess complicating diverticulitis,
appendicitis, and other disease that
alter the integrity of the gastrointestinal
tract
b. S. faecium / E. faecium
c. S. durans / E. durans
E. Group E – no known representative
F. Group F (S. anginosus)
G. Group G (S. canis)
H. Group H (S. sanguis)
I. Group K (S. salivarius)
J. Group N (S. lactis)
Streptococcus pyogenes
 _____________________
 metabolism is ____________ (lactic acid:
principal product)
 catalase negative
 oxidase negative
 does not contain heme compounds
 killed in 30 minutes at 60oC
Cultural Characteristics:
 optimal at 7.4 to 7.6 at 37oC
 growth is enhanced at a reduced oxygen
tension or ___________________
 _______________ on sheep blood agar
 colonies are domed, grayish to opalescent
and approximately 0.5mm in diameter
Determinants of Pathogenicity:
1. Lipoprotein – mediate the adherence to buccal
epithelial cells
2. _____________ – the major virulence factor of
Group A streptococci; acid and heat stable;
trypsin sensitive
3. Hemolysins
S. pyogenes – responsible for the nonsuppurative
postinfectious sequelae of ______________
and ______________________
Acute Streptococcal Infection
1. Pharyngitis
2. Impetigo / Streptococcal pyoderma – disease
of temperate climates and occurs with highest
frequency in late summer and early fall;
organisms first colonize normal skin and
secondary appear in the pharynx
Clinical Manifestations:
1. ________________________
2. Skin Infection – syndromes of impetigo,
cellulitis, erysipelas, wound infection and
gangrene
a. _________ – very superficial infection that
usually begins a small vesicles,
progressing to weeping lesions with an
amber crust and slightly cloudy purulent
exudates
b. Cellulitis with lymphangitis and
lymphadenitis after deeper invasion of
streptococci
c. _____________ – an infection of the skin
and subcutaneous tissues that usually
occurs on the face or the lower extremities.
The lesion is characterized by erythema,
edema and induration, with a distinct
advancing border.
3. ______________________ – common cause
of maternal mortality
Sequelae of Acute of Streptococcal Infection
1. _____________________________________
RF is a nonsuppurative inflammatory
reaction which occurs only after pharyngitis. It
is manifested by arthritis, carditis, chorea,
erythema marginatum, or subcutaneous
nodules. These symptoms usually occur within
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 2 to 3 weeks of the onset of streptococcal
infection.
2. _____________________________________
AGN is a post-infectious complication
which may be seen after either pharyngeal or
cutaneous infection. It is most often seen in
children and may be associated with the acute
onset of edema, oliguria, hypertension,
congestive heart failure, or seizures.
Streptococcus agalactiae
 tend to grow as _______ or in short chains
 colonies are usually large and mucoid with
small zone of β hemolysis
 5-15% of isolates may be non-hemolytic
 97% of strains produce yellow, red or
orange pigment
 positive _________________
 characterized by an accentuated
zone of hemolysis when the group
B streptococcus is inoculated
 majority grow ________________; few
may grow in the presence of __________
 Esculin is not hydrolyzed.
 small percentage are bacitracin sensitive
 produces DNAses, hippuricase,
neuraminidase and hyaluronidase
Other Streptococcal Infections:
____________________
 implantation of bacteria or fungi on the
endocardial surface of the heart
 most frequently encountered on damaged
valves, in congenital heart disease or
prosthetic valves
Miscellaneous:
 Pneumonia, septic arthritis, biliary or intra-
abdominal infection, urinary tract infection,
meningitis, osteomyelitis, or sinusitis
Enzymes and Toxins Produced by Streptococci:
1. Erythrogenic Toxin – fever-causing toxin
_____________ – form of streptococcal
pharyngitis by a strain of S. pyogenes refer to
as S. scarlatinae
a. __________
 susceptibility test for scarlet fever on
the arm of the patient
Inject 0.1cc of toxin on the test arm.
Inject 0.1cc of toxoid on the control arm.
(+) : ____________________
Test arm containing toxin is positive for
scarlet fever.
Control arm containing toxoid is negative to
scarlet fever.
b. __________________________________
 a test to check if the skin rashes are due
to scarlet fever or not
 inject into area of rashes an
antierythrogenic toxin. Is skin rashes
fade or blanch, it is due to scarlet fever.
Principle:
Based on the neutralization of
erythrogenic toxin when an antitoxin is
injected into the test arm of the patient
suspected of having scarlet fever.
2. Leucocidin
3. Streptodornase – an enzyme used to liquefy
exudates
4. Hemolysin
5. Hyaluronidase / Spreading factor
6. Streptolysin – responsible for hemolytic
reaction
a. ________________ – oxygen labile,
antigenic toxin, responsible for subsurface
hemolysis
b. ________________ – oxygen stable, non-
antigenic toxin, responsible for surface
hemolysis
Laboratory Diagnosis
1. G/S – to demonstrate gram (+) cocci
occurring singly but generally in chains
2. Culture the microorganisms and prefer the
BAP.
________________ – with inhibitory
component that inhibits the growth of H.
hemolyticus (normal throat commensal
whose colonies can be mistaken to the
colonies of beta-hemolytic streptococci)
3. Perform the following tests:
a. __________________
b. __________________
c. ___________________
d. ______________
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 e. ________________ 4. Optochin Disc
4. Serological test Test
5. Quinidine Test
a. ASO Titer 6. Animal
b. Gel diffusion Inoculation Test
c. Slide and Capillary Agglutination test (Mouse
d. Counter Immuno Electrophoresis Virulence)

Streptococcus pneumoniae Identification Tests for Pneumococci:

 Diplococcus pneumoniae / Pneumococcus 1. Bile Solubility Test
 gram (+) cocci Bile acts upon the cell wall of pneumococci
 ________________ in shape (lanceolatus) bringing about lysis. This test is based on the
 the most common cause of bacterial presence in pneumococci of an autolytic
pneumonia / ___________________ amidase which is activated by bile leading to
 important cause of otitis media, meningitis the dissolution of organism.
and septicemia
Principle:
Cultural Characteristics: Based on the presence of the enzyme
 ____________________________ autolytic amidase, which causes the lysis of the
 facultative anaerobic requiring _________ organism.
for growth
 require ____________ (nitrogenous 2. Optochin Disc Sensitivity Test
alcohol) for nutrition  test most widely used for the presumptive
 BAP: ________________ identification of the pneumococcus
Young colonies – circular, glistening and  primarily designed to distinguish
dome-shaped pneumococci from viridians streptococci
Aged colonies – colonies become  ______________ –
flatted with raised margin and depressed ethylhydrocupreinhydrochloride; a quinine
centers, giving rise to doughnut shape, derivative that inhibits the growth of
umbilicate, nail head, checker colonies pneumococci but not of viridians
streptococci
 at BAP, heavily inoculated with suspected
Two Substances Responsible for Serological organism, place Optochin disc, incubate at
Reaction: 35-37oC for 24 hours
1. Specific Soluble Substance (SSS) – a (+) : _____________________
polysaccharide capsular substance which _______ = zone of growth of inhibition
determines both the virulence and capsular depending on the size of
types the Optochin disc used
2. Somatic Antigen – referred to as C- 6mm disc = 14 mm
polysaccharide present in the cell wall; C- 10mm disc = 16mm
polysaccharide binds with C-reactive
protein 3. Capsular Precipitation Test / Neufeld-
Quellung Reaction
Test Differentiating Pneumococci from  the most useful and rapid method for the
Other Streptococci: identification of S. pneumoniae
 the most accurate, reliable, specific test
Test Pneumococci Other Strep  to a freshly emulsified sputum, add a
1. Bile Solubility loopful of antipneumococcal serum and
Test methylene blue
2. Inulin  essentially Ag-Ab reaction
Fermentation
Test
(+): ______________________________
3. Capsular __________________________________
Precipitation
Test

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4. Animal Inoculation Test / Mouse Virulence
Test
 White mouse is particularly susceptible to
small inocula of pneumococci, such that if
we inject sputum intraperitoneally into the
white mouse, the animal dies within 16 to
48 hours. Upon the death of animal, extract
the heart blood and culture it. We can get a
pure culture of pneumococci.
5. Francis Test
 __________ to determine the presence or
absence of antibody against pneumococci

Clinical Manifestations:
1. Pneumonia – strikes with a single violent
shaking chill and fever that ranges between
102-106oF.
Complications: pleural effusion empyema
Meningitis, pericarditis & endocarditis

2. Upper Respiratory Tract Infections

S. pneumoniae – leading bacterial pathogen in
infants and children and accounts for a large
number of infections of the respiratory tract and
adjacent structures E.g. middle ear

3. Extrapulmonary Infections
_____________ – the most serious
pneumococcal infection

Other Gram (+) Cocci:

Gaffkya tetragena / M. tetragena –
characteristically arranged in packets of 4 or
tetrads

Sarcina lutea / Saprophytic micrococci – occurs

in cubical packets of 8’s, 16’s, 32’s or more;
produce bright yellow pigments

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  separated from Neisseria due to
_____________ and due to advent
hydrogenation
 lactamase (+)
 capsulated contributing to its
invasiveness
 ferments _______________________
 normal oral and nasopharyngeal flora

CHO Fermentation Test

Glucose Maltose Sucrose Lactose
M. catarrhalis
N. gonorrheae
N. meningitidis
N. subflava
N. lactamica
N. sicca
N. flavescens
N. sicca is also a fructose fermenter.

Other Gram-Negative cocci:

Veilonella parvula
 small, strictly anaerobic organism with the
production of fatty acid end products like
propionic and polypeptide

Morococcus cerebrosus
 gram (-), oxidase (+) coccus isolated from
the brain abscess