GRAM POSITIVE COCCI

Gram positive cocci

Micrococcacaea Streptococcus spp.

S. pyogenes
S. agalactiae
S. mitis
Enterococci
S. pnuemoniae
Staphylococcus spp

S. aureus
S. epidermidis
S. saprophyticus
Micrococcus
Catalase test
Reagent used: 3% hydrogen peroxide (H202)
Positive result: effervescence (bubbling)
Micrococcaceae: positive (+)
Streptococcus spp: negative (-)
STAPHYLOCOCCI MICROCOCCI
BACITRACIN/TAXO A DISK TEST Resistant Susceptible
FURAZOLIDONE SUSCEPTIBILITY TEST Susceptible Resistant
MODIFIED OXIDASE/MICRODASE TEST Negative Positive
LYSOSTAPHIN SENSITIVITY TEST Sensitive Resistant
GROWTH ON FUROXONE-TWEEN 80-OIL
RED O AGAR
Negative Positive
ACID PRODUCTION FROM GLYCEROL Positive Negative
OXIDATION-FERMENTATION REACTION
Genus name is derived form greek work staphle meaning bunches of
grapes
Gram positive cocci in clumps
Belongs to family of Micrococcaceae
non-motile, non-spore forming and glucose fermenter

Most virulent species of Staphylococci
-hemolytic on BAP
Halophilic (can grow in presence of 7.5-10% NaCl)
Produces a golden yellow pigment (staphyloxanthin)
Diseases and infections:
Toxin-Induced diseases: food poisoning; scalded skin syndrome (SSS) or
ritters disease; toxic shock syndrome (TSS)
UTI
Acute bacterial endocarditis
Cutaneous infections (folliculitis, furuncles/boil, carbuncles, impetigo)

Virulence factors:
Catalase
A heme enzyme that catalyzes the decomposition of H202
H2O2 H2O + O2

Coagulase
Coagulates the fibrinogen to fibrin
Promotes formation of fibrin layer around staphylococcal abscess thereby protecting the
bacteria from phagocytosis\
2 types
1. cell-bound coagulase/clumping factor
2. Unbound coagulase/free coagulase

Hyaluronidase (spreading factor enzyme)
Enhances invasion and survival in tissue; breaks down hyaluronic acid present in the
intracellular ground substance of connective tissues resulting to spread of bacteria


Virulence factors:
Staphylokinase
Fibrinolytic activity (dissolves fibrin)

Lipase (fat-splitting enzyme)
Essential for survival in sebaceous areas of the body

DNAse and phosphatase
Destroys DNA

-lactamase
It breaks down penicillin and other -lactam drugs

Enterotoxins (heat stable)
Acts as neurotoxins that stimulate vomiting



Virulence factors:
Enterotoxin F
Stimulates production of large amount of cytokines and causes almost all cases of
menstruating-associated TSS

Leukocidins/panton-valentine leukocidin (cytolytic toxin)
Attacks and kills the WBC and prevents phagocytosis

Hemolysin (cytolytic toxin)
Causes anemia make iron available for bacterial growth

Exfoliatin A and B
Causes the epidermal layer to slough off

Protein A
Antiphagocytic by competing with neutrophils for the Fc portion of opsonins




Differential tests for S. aureus
Coagulase test
2 methods:
1. Slide method
- screening test
- detects cell bound coagulase/clumping factor
- (+) result: clot/coagulum formation
- other slide coagulase positive organisms: S. lugdunensis, S. schleiferi
2. Tube method
- definitive test
- detects free/unbound coagulase
- procedure: inoculate a tube containing plasma and incubate at 35C for 1-4 hours
- (+) result: clot/coagulum formation
- if no clot appears after 4hrs, incubate the tube at room temperature for 20 hours
-other tube positive coagulase organisms: S. hyicus, S. intermedius, S. delphini




Differential tests for S. aureus
Mannitol Salt Agar:
Contains 1% mannitol + 7.5% NaCl
pH indicator: phenol red
(+) S. aureus: colonies surrounded by yellow halo

Tellurite Glycine Agar:
S. aureus: jet black colonies

Polymyxin sensitivity test:
S. aureus: resistant
Other Staphylococcus spp.: sensitive






Differential tests for S. aureus
Voges-Proskauer test
It diffentiates S. aureus from S. Intermedius
(+) pink color: S. aureus

DNAse Test
Medium: DNA medium
Reagent: methyl green
(+) result: clearing of the dye









Normal flora of skin
Common contaminant of medical equipments (prosthatic heart valve
implants, catheters)
Secretes poly-gamma-DL-glutamic acid: provides adherence to devices
Common nosocomial infections
Non hemolytic on BAP
MSA (-), coagulase (-)
Novobiocin susceptible








Associated with community-acquired UTI in young, sexually active
females
Non hemolytic on BAP
Coagulase (-), MSA (-)
Novobiocin resistant







Belong to family Streptococcaceae
Commonly found as part of human normal flora but can cause life
threating infection once they invade normally sterile sites of the body
Gram positive cocci in chains or pairs
Facultative anaerobes; some species are capnophilic








Classification of Streptococci
1. Academic/Bergeys Classification
Based on temperature requirment

2. Smith and Brown Classification
Based on hemolytic patterns

3. Lancefield Classification (antigen serogrouping)
Based on extraction of C carbohydrate from the streptococcal cell wall
Mostly significant in classifying and identifying -hemolytic streptococci








Academic/Bergeys Classification









PYOGENIC
GROUP
VIRIDANS
GROUP
LACTIC GROUP ENTEROCOCCUS
GROUP
Temperature
requirement
37C Can grow at 45C
and 37C

Can grow at 10C
and 37C

Can grow at 10C,
45C and 37C
Type of
hemolysis on
BAP
-hemolytic May be or -
hemolytic
-hemolytic

May be , , -
hemolytic
Other
information
Produces pus Normal flora of
the URT of
humans
Often found in
dairy products
Normal flora of
the human
intestine
Species S. pyogenes S. salivarius, S.
mutans, S. mitis
S lactis E. faecalis
Smith and Brown Classification
Alpha-hemolytic
S. pneumoniae

Beta-hemolytic
S. pyogenes, S. agalactiae, S. dysagalactia subsp. Equimilis and s. anginosus group

Gamma-hemolytic
E. fecalis







Lancefield Classification
Group A Streptococci
Pathogenic to man
Acquired thru contaminated droplets by cough or sneeze
Colonies are small, translucent and smooth; well-defined -hemolytic
Species: S. pyogenes (fever-producing bacteria; flesh-eating bacteria
involves deeper tissue and organs)




Group A Streptococci
Virulence Factors:
1. M-protein
- principal virulence factor
- attached to the peptidoglycan
- antiphagocytic
- for adherence to mucosal cells
2. Protein F
- mediates epithelial cell attachment
3. Lipoteichoic acid
- bacterial adherence to the respiratory epithelium
4. Hyaluronic acid and capsule
- weakly immunogenic
-prevents opsonized phagocytosis
- masks its antigen





Group A Streptococci
Virulence Factors:
4. Hemolysins
a. Streptolysin O (oxygen labile)
-responsible for subsurface hemolysis
- highly antigenic
b. Streptolysin S (oxygen stable)
- responsible for surface hemolysis
- non antigenic
5. DNAse
- 4 types: A, B, C and D (antigenic enzymes)
- it lowers viscocity of exudates
6. Streptokinase
- it cause lysis of fibrin clots
- allows the bacteria to move from the clotted area (spread of
infection)





Group A Streptococci
Virulence Factors:
7. Hyaluronidase
- Spreading factor

8. Pyrogenic toxins
- super antigens
- formerly known as erythrogenic toxin
- exotoxin B- degrades proteins; mediates rash in scarlet fever




Group A Streptococci
Infections and Diseases:
1. Pharyngitis/tonsilitis (strep throat)
2. Scarlet Fever (Scarlatina)
- Caused by the release of pyrogenic exotoxins
- Cardinal signs: diffused red rash on the upper chest to the trunk and to the
extremities and strawberry red tongue
- Susceptibility Test: Dicks test
- (+) result: erythema or redness of the test site
- Diagnostic test: Schultz Charlton
- (+) result: blanching phenomenon rash fade
3. skin infections
a. cellulitis
b. impetigo
c. erysipelas


Group A Streptococci
Infections and Diseases:
4. Rheumatic Fever
- characterized by fever, inflammation of heart, joints and
blood vessels

5. Acute Glumerulonephritis (AGN) or Brights disease
- inflammatory disease of the renal glomeruli, results from
deposition of antigen-antibody complexes in the kidneys

6. Streptococcal TSS
- a condition which the entire organ system shuts down

Group A Streptococci
Lab diagnostic tests:
1. Bacitracin Disc Test/ Taxo A
- differentiates S. pyogenes from other -hemolytic Strep
- (+) result: Group A- susceptible

2. Sulfamethoxazole and trimethoprim (SXT) test
- (+) result: Group A- resistant

3. L-pyrolidonyl--naphthylamide (PYR) test
- detects pyrolidonyl-arylamidase enzyme
-(+) result: bright/cherry red color
Lancefield Classification
Group B Streptococci
Part of the normal flora of female genital tract and lower GIT
Can cause infections to fetus during passage through the birth canal
Grayish white, mucoid colonies with small zone of -hemolysis
Species: S. agalactiae
Infections and Diseases:
Pneumonia
Meningitis
Neonatal sepsis
Postpartum infection
Osteomyelitis
UTI
endocarditis




Group B Streptococci
Lab diagnostic tests:
1. CAMP Test (Christie, Atkins, and Munch-Peterson test)
- differentiates S. agalactiae from other -hemolytic strep
- (+) result: arrow head -hemolysis near growth or bowtie
appearance

Group B Streptococci
Lab diagnostic tests:
2. Hippurate Hydrolysis Test
- reagents: sodium hippurate + ninhydrin
- (+) result: S. agalactiae- purple color after ninhydrin rgt
Lancefield Classification
Group C Streptococci
Organisms are recovered from URT, vagina and skin of humans
Possess M protein just like the group A streptococci
Animal pathogens
Species: S. dysagalactiae subsp equisimilis, S. equi subsp. zooepidermicus




Lancefield Classification
Group D Streptococci (S. bovis/Nonenterococci)
- produces alpha-prime hemolysis
- S. bovis is no longer a valid species name; based on DNA
studies, S. equinis and S. bovis are the same
- often isolated in blood cultures of individuals with GI
carcinoma


Belong to family streptococcaceae
Formely known as group D streptococci
Natural inhabitants of the intestinal tracts of humans and animals
Not highly pathogenic but can cause nosocomial infections
Can grow in extreme conditions (alkaline pH, can grow at 45C and salt
solution
Can exhibit , , - hemolysis
Species: E. fecalis (most common), E. faecium, E. avium, E. gallinarum, E.
durans, E. raffinosus

ENTEROCOCCUS NONENTEROCOCCUS
Bile esculin hydrolysis (+) black color complex (+) black color complex

PYR test + -
Growth in 6.5% NaCl + -
Penicillin susceptibility test Resistant Susceptible
TESTS TO DIFFERENTIATE GROUP D NONENTEROCOCCUS FROM ENTEROCOCCUS SPP
Aka Diplococci/pneumococci
Considered normal flora of 25-50% of the URT of children
Most common cause of bacterial meningitis in adults
Gram positive cocci in pairs, oval or lancet shape
Young colonies: alpha hemolytic mucoid, dome-shaped glistening colonies
Old colonies: alpha hemolytic mucoid, flat colonies with depressed center
(nail-head appearance)
Principal virulence factor: capsular polysaccharide
Infections and diseases:
1. Lobar pneumonia
- shows bloody, rust-tinged sputum
- Results from the disturbance of the normal defense barriers
- Predisposing factors: alcoholism, anesthesia, malnutrition
2. Meningitis
3. Otitis media
4. Bacteremia
5. Endocarditis
6. peritonits
S. PNUEMONIAE VIRIDANS GROUP
Mouse virulence + -
Inulin fermentation + -
Bile solubility (+) soluble -
Optochin/Taxo
P/Ethylhydrocupreine
hydrochloride
Sensitive Resistant
Neufeld Quellang (capsular
antibodies)
+ -
TESTS TO DIFFERENTIATE S. PNEUMONIAE FROM VIRIDANS GROUP
Formerly known as nutritionally variant streptococci (NVS)
Pyridoxal (vit B12) dependent, thiol-dependent (cystein) and symbiotic
streptococci
Part of human oral and GI normal flora
Opportunistic pathogens of low virulence
Gram variable and pleomorphic forms
Appears satellite around an organism that produces pyridoxal (E. coli,
Klebsiella spp, Enterobacter spp and yeast)
Aerococcus
- Common airborne bacterium
- Resembles as viridans streptococci on culture but similar to
staphylococci microscopically
- May show pseudocatalase reaction
- Halophilic
- Species: A. viridans bile esculin and PYR (+)
A. urinae bile esculin and PYR (-)
Gemella
- Similar to colony morphology and habitat of viridans group
- Easily decolorize on gram stain (may appear gram negative cocci in
pairs, tetrads, clusters or short chains
- Species: G. haemolysans, G. morbiliorum, G. bergeriae and G. sanguinis
Lactococcus
- Previously classified as group N streptococci
- Physiologically similar to enterococci but do not produce acid from
carbohydrates
Leuconostoc
- They have irregular coccoid morphology
- Share several phenotypic and biochemical characteristics with
Lactobacillus, viridans Streptococci, Pediococcus and Enterococcus
- Species: L. citreum, L. cremoris, L. dextranicum, L. lactis
Pediococcus
- Can be misidentified as viridans streptococci or enterococci
- Grow at 45C
- Isolated from individuals who have GI abnormalities or have undergone
GI surgery
S.
pyogenes
S.
agalactiae
S.
Pneumoniae
Viridans
streptococci
Grp D
streptococci
Enterococci
Bacitracin S R S R R R
SXT R R S S V R
Optochin R R S R R R
Hippurate
Hydrolysis
- + - - - -
CAMP - + - - - -
PYR + - - - - +
LAP + + + + + +
Bile Esculin - - - - + +
Growth in
6.5% NaCl
- - - - - +
SUMMARY OF BIOCHEMICAL TESTS OF STREPTOCOCCI