Gram positive cocci

Family: Micrococcaceae
Micrococcus
gm (+) tetrads or cuboidal packages; catalase (-); coagulase (+); NM;
on BAP gamma haemolytic; grows on 5% but not on 7.5% NaCl;
Oxidase Test (+) blue
Lysostaphin and Furazolidone (R)

Planococcus not associated with human infections

Gaffkya tetragena arranged in tetrads; non-pathogenic; found in respiratory
tract;
Sarcinae large gm (+) cocci; cuboidal packages; can be cultivated from
stomach contents
Staphylococci - gram (+) organisms in grape like clusters
facultative anaerobes, NM, NS, NE; Strongly catalase (+); Modified
Oxidase (-)
Lysostaphin and Furazolidone (S)
Bacitracin resistant; Grows in 7.5 10% NaCl; ferment glucose; buttery
looking
As to pigment formation/ growth on Loefflers serum slant (LSS)
S. aureus golden yellow
S. citreus lemon yellow
S . albus - white pigment
Staphylococcus aureus
Facultative anaerobes; gr (+) cocci in clusters; golden grape-cluster berry
Cultural Characteristics:
On agar: smooth, opaque and circular colonies
On broth: produce uniform turbidity
On BAP: beta-hemolytic
On Tellurite agar: produces jet black colonies
Produces golden yellow pigment
Catalase (+); strongly coagulase (+);
Ferment mannitol ; Phosphatase (+); ONPG (+); Arginine (+); Gelatin (+); VP
(+)
Nitrate and Dnase (+); PYR (-); Lysostaphin and Novobiocin sensitive;
Polymixin resistant
pseudocatalase reaction Enterococcus, Rothia (formerly) Stomatococcus and
Aerococcus

Virulence factors/toxins:
- Catalase decomposition of hydrogen peroxide to water and oxygen
- Hyaluronidase breaks down hyaluronic acid in between cells resulting to
spread of bacteria
- Protease
- Coagulase
- Lipases sebaceous areas of the body (boils, furuncles)
- Staphylokinase dissolves the fibrin clot
activates plasminogen to form plasmin, which digest fibrin clots
- Deoxyribonuclease destroys DNA with the help of phosphatases; lowers
viscosity of exudates;
- Urease
- Leukocidin also known as Panton-Valentine Leukocidin (PVL)
(PMN, macrophage and monocytes) cytolytic sporeforming exotoxins that kills
WBC; suppresses phagocytosis
- beta-lactamase most of staphylococci isolates are R to PG Clumping factor
clumping factor A
- Exfoliatin A and B
stratum granulosum (surface layer of the skin) is destroyed
- S. aureus beta-toxin
- Hemolysin cytolytic toxins causing anemia
alpha toxin destroys RBC, platelets, skin destruction
beta toxin destroys sphingomyelin and RBC around nerves (hot-cold lysin)
gamma toxin injury in RBC produces edematous lesions
delta toxin destroys RBC
- Enterotoxins - heat stable; low molecular weight; water soluble
Enterotoxin A, B, C1, C2,D,E, and F
For food poisoning (A and D) cause diarrhea, vomiting among humans
For pseudomembranous enterocolitis (B)
Enterotoxin F and exotoxin type C associated with TSS
E. coli 0157:H7 / Clostridium difficile / Clostridium perfringens / Vibrio cholera
(cholera toxin)
Yersinia enterocolitica / Shigella dysenteriae (Shiga toxin)
Viral: Rotavirus (NSP4)
- Pyogenic exotoxin associated with scarlantine syndrome; causes TSS among
females
- Enterotoxin F causes toxic shock syndrome
- Protein A active
- substance found in the cell wall

Differential Tests for Staphylococcus aureus

1. Coagulase Test:
Slide Coagulase test:
Principle: Also known as the clumping factor; converts the alpha and beta
fibrinogen into fibrin in plasma that deposits on the cell wall. As a result
coccus stick to each other and clumping is observed.
determines the bound coagulase cell wall associated protein; reacts with fibrinogen
to form a coagulate
Procedure:
1. 2 gtts of saline on slide then emulsify staph colonies
2. Add a drop of rabbits plasma and mix with wooden applicator stick
3. Macroscopic clumping of plasma within 10-30 seconds is a positive result.
Other slide coagulase + organisms: Staphylococcus lugdunensis and
Staphylococcus schleiferi
Perform tube coagulase test for confirmation
*any negative slide test should be confirmed with the tube method
(+) result - clot/coagulum formation within 30 seconds
Other slide coagulase (+) organisms: S. lugdenensis / S. schleiferi
Tube Coagulase test:
Principle: Free coagulase secreted by S. aureus reacts with coagulase
reacting factor (crf) in plasma to form thrombin, this converts fibrinogen to
fibrin resulting in clotting of plasma
Extracellular enzyme;
determines the free coagulase secreted extracellularly which reacts with prothrombin
and its derivatives
clot formation within 1-1/2 hours or within 4 hours (+) result
indicative of virulent Staphylococcus aureus
absence of coagulation after 24 hrs of incubation = (-) result
citrate utilizing organisms may yield false + results
Other tube coagulase (+) organisms: S. hyicus (variable ) / S. intermedius
(strongly +)
Citrate utilizing organisms may yield false (+) results: plasma containing EDTA
rather than citrate should be used
2. Mannitol Fermentation test
MSA (inhibitor - 7.5% salt) is both selective and differential
pH indicator phenol red / acid yellow alkaline red
positive yellow color
some strains of S. saprophyticus ferments mannitol
S. saprophyticus - also ferments mannitol

3. Bacitracin/Taxo A Disk Test

Differentiates staphylococci from micrococci
0.04 units bacitracin performed on BAP
Results: Micrococci susceptible (10 mm)
Staphylococci resistant
4. Furazolidone Susceptibility Test
Differentiates staph from micrococci
100 ug furazolidone performed on BAP
Results: Staph susceptible (15 mm); Micrococcus resistant
5. Modified Oxidase/Microdase Test
Differentiates staph from micrococci
Reagent paper: tetramethyl-p-phenylenediamine dimethylsulfoxide
(+) blue color within 2 minutes Micrococci
6. Lysostaphin Sensitivity Test
(+) 10-16 mm zone of inhibition S. aureus (sensitive); Micrococci (resistant)
S. aureus lysed with lysostaphin
7. Tellurite Glycine Agar
(+) jet black colonies
8. Polymixin Sensitivity Test
(+) result: resistant S. aureus
9. Voges-Proskauer
Result: S. aureus (+) pink (production of acetyl methyl carbinol/acetoin)
S. intermedius (-)
Reagent: 40% KOH + 1% alpha naphthol
S. aureus is sensitive to lysostaphin, novobiocin; Resistant to polymixin
Positive for nitrate, VP, ONPG, DNASE test
Detects DNASE in two methods:
a. Dye method
1. DNA + methyl green = clear zone around the colonies
2. DNA + toluidine blue = pink zone around the toluidine blue
b. HCl precipitation method
Other DNASE positive: S. aureus, Moraxella, Serratia
(+) result: clearing of agar
S. lugdunensis confused with S. aureus; may cause catheterilated bacteremia;
ferments mannitol; slide coagulase (+); PYR (+)
Pathogenecity
a) Skin infections : boils or furuncles, carbuncles, acne, endocarditis, impetigo
boil, also called a furuncle, is a deep folliculitis, infection of the hair follicle
Cellulitis is a diffuse inflammation of connective tissue with severe
inflammation of dermal and subcutaneous layers of the skin
A carbuncle is usually with one or more openings draining pus onto the skin

b) Toxin mediated diseases: Food poisoning; scalded skin syndrome and toxic
shock syndrome

Staphylococcal scalded skin syndrome, SSSS, or Localized bullous

impetigo is a dermatological condition caused by Staphylococcus
aureus.
The syndrome is induced by epidermolytic exotoxins Aand B, which are
released by S. aureus and cause detachment within the epidermal layer

disease presents with the widespread formation of fluid filled blisters that are
thin walled and easily ruptured.

Culture must be 24-36 hours.

Staphylococcus citreus lemon yellow pigment
Staphylococcus albus porcelain white pigment
A positive coagulase test is the only single criterion used for ID of
Staphylococcus aureus
Staphylococcus hyicus and Staphylococcus intermedius produced coagulase (+);
MRSA people who had recent surgery or a hospital stay have an increased risk of
developing MRSA.
Also seen in older people, nursing homes and weakened immune system
MRSA strains have recently emerged outside the hospital becoming known as CAMRSA or superbug strains of the organism, which now account for the majority of
staphylococcal infections seen in the ER or clinic.
S. aureus is a major cause of hospital acquired (nosocomial) infection of surgical
wounds and infections associated with indwelling medical devices. S. aureus causes
food poisoning by releasing enterotoxins into food, and toxic shock syndrome by
release of superantigens into the blood stream.
Staphylococcus epidermidis
Gram (+) cocci in clusters; white, opaque pinhead colonies
Coagulase (-), does not ferment mannitol; Sensitive to novobiocin
Non-hemolytic on BAP; nonpigmented Dnase (-); MSA (-)
may cause bacteremia, UTI, stitch abscess; meningitis and endocarditis
Virulence factor -exopolysaccharide that adhere on agar surface
Contaminants on catheterization instruments and heart valve implantation
Staphylococci saprophyticus

coagulase (-); does not ferment mannitol; white, opaque, pin head slightly
larger colonies

Resistant to novobiocin and nalidixic acid; MSA (+)

Dnase (-) and non-hemolytic on BAP
Most common cause of mild and acute UTI May cause pyelonephritis , cystitis
and bacteriuria
Novobiocin sensitivity - differentiate S. epidermidis from S. saprophyticus

*Resistance of Staph to penicillinase-resistant penicillin (methicillin, oxacillin and

nafcillin) is due to mecA gene which encodes an altered penicillin binding protein
known as PBP2a.

Tests to differentiate STAPHYLOCOCCI from MICROCOCCUS

STAPHYLOCOCCI
MICROCOCCI
Lyostaphin Susceptibility
S
R
Modified Oxidase Test
Negative
Positive
Rgt paper: tetramethyl-p-phenylenediamine dimethylsulfoxide) + result =
blue color w/in 2 mins
Bacitracin Susceptibility
R
S
(0.04 units bacitracin on BAP) taxo A
Furazolidone Susceptibility
S
R
(100 ug furazolidone on BAP)

Catalase test
Coagulase test
Mannitol Fermentation
Hemolysis on BAP
Novobiocin
DNASE test
Others
cause of UTI in young

S. epidermidis
S. saprophyticus
(+)
(+)
(-)
(-)
(-)
(-)
(-) gamma
(-) gamma
S more than 16 mm
R less than 16 mm
(-)
(-)
normal skin flora, causes
most common
UTI, stitch abscess

women, causes pyelonephritis

and
Prosthetic heart valve inf.
indwelling catheter
Laboratory Diagnosis:
1. Gram stain

cystitis in those with

2.
3.
4.
5.
6.

Culture on BAP, PEA, CAP, Fluid Thioglycollate, BHI

Catalase test
Coagulase test
Mannitol fermentation test
DNASE test
a. 0.1 N HCl agglutinate or precipitate protein
b. 0.1% toluidine blue (+) pink color cell died / (-) blue color cell is alive
7. Phage typing
8. Pulse Field Gel electrophoresis
9. Novobiocin
10.Modified Oxidase Test rgt: tetramethyl-p-phenylenediamine dihydrochloride
(+) blue/purple Micrococcus luteus
(-) no color change (S. aureus)

Test to differentiate Staph and Micrococci

Staph
Aerobic growth
(+)
Anaerobic growth
(+)
Lyostaphin Susceptible
S
Modified Oxidase Test
(-)
Bacitracin
R
Catalase
(+)
Glucose utilization
Fermenter

Micrococci
(+)
(-)
R
(+)
S
(+)
Oxidizer

Stomatococcus mucilanginosus emerging pathogen of an immunocompromised

patient
Known to cause endocarditis, bacteremia; weakly catalase (+); coagulase (-);
unable to grow in 5%NaCl
Modified Oxidase (-); Lysostaphin and Furazolidone (R)
Streptococci
General Characteristics:

Gram (+) spherical to ovoid , in chains/pairs

NM, NS, NE; Catalase (-) and Oxidase (-)

Many are facultative anaerobes and capnophilic

Insoluble in bile; does not ferment inulin; Susceptible to vancomycin

With tiny, discrete, mucoid slightly opaque to grayish pinpoint translucent

colonies on BAP

Young cultures are characterized by the presence of capsule

Growth is enhanced by blood, serum or glucose

All streptococci except the viridans group have a layer of C carbohydrate

serological classification

Colonial morphology: hemolysis on 5% sheeps blood

The following organisms resemble streptococci: Leuconostoc, Lactococcus,
Aerococcus, Pediococcus
Gemella, Enterococcus, Aloiococcus, Globicatella
Vancomycin Resistant Test differentiate Pediococcus from Strep. viridans
Colonies:
- Mucoid (capsule) /rough or matte (large amt of M protein)/smooth or glossy
(little amt of M protein)
Streptococci are serotyped using known antibodies to the:
Capsules for S. pneumonia / M-protein for S. pyogenes
Smith and Brown Classification
1. Beta-hemolytic - Streptococcus pyogenes; S. agalactiae, Groups C, F and G ;
complete and clear zone of hemolysis
2. Alpha-hemolytic Streptococcus pneumonia; Viridans Streptococci (S. mitis
and S. mutans); partial or incomplete hemolysis; greenish
Note: alpha prime a small zone of alpha hemolysis surrounded by zone of beta
hemolysis after refrigeration
3. Gamma-hemolytic/Nonhemolytic Enterococcus faecalis (Group D); E. faecium;
S. bovis (Group D)
Academic or Bergeys Classification
1. Pyogenic - S. pyogenes; will not grow on neither 10C or 45C only at 37C;
produce pus
2. Viridans - S. mitis, S. mutans, S. sanguis, S. salivarius , S. constellatus, S.
intermedius
will grow at 45C and 37C
3. Enterococci - E. faecalis; will grow both at 10C and 45C and 37C
4. Lactic group - S. lactis and S. cremoris; will grow on 10C and 37C
Lancefield Classification
Rebecca Lancefield found out that C CHO can be extracted from the streptococcal
cell wall by placing the organisms in dilute acid and heating for 10 minutes
1. Group A ( Streptococcus pyogenes ) pathogenic to man; airborne
other virulence factors (Protein F mediates cell attachment/ Lipoteichoic
acid bacterial adherence to the respiratory epithelium); not considered part
of the normal flora
Enzymes and Toxins produced by S. pyogenes
1. Erythrogenic toxin(pyrogenic toxin) serotypes A, B, C
Exotoxin B (cysteine protease) degrades proteins; mediates scarlet
fever; only toxin that is produced by S. pyogenes
2. Hemolysins

Streptolysin O - highly antigenic and O2 labile; cause lysis of WBC,

platelets and tissue cells; Streptolysin S O2 stable; non-antigenic
Cause lysis of WBC
3. Deoxyribonucleases
4. Streptokinases protein that binds to plasminogen and activates
production of plasmin; allows the bacteria to move from the clotted area
5. Hyaluronidase separate the tissue and spread the organism
Infections and Diseases:
1. tonsilitis/pharyngitis
2. scarlet fever (scarletina):
cardinal signs: red (erythema) rash (exanthem) on the upper chest and
spreads to the trunk and extremities
DIAGNOSTIC TEST FOR SCARLET FEVER
A) Dicks Test
(+) erythema - redness
B) Shultz Charlton test diagnostic test
(Blanche Phenomenon)
3. Skin Infections - erysipelas or St. Anthonys fire, cellulitis, acute
glomerulonephritis, galloping gangrene, impetigo
Cellulitis spreading infection of subcutaneous skin tissue; accumulation of fluid
Erysipelas inflammation of the dermal layer of the skin characterized by painful
reddish patches
4. Rheumatic fever - disease affecting joints and heart valves
- ASO Test
5. Acute glomerulonephritis or Brights disease inflammatory disease of renal
glomeruli
6. Others: Wound, burn, pyoderma
TEST FOR GROUP A BETA HEMOLYTIC STREPTOCOCCI / Diagnostic Tests
a) Bacitracin Disk Test (0.04 units /Taxo A) to diff S. pyogenes from other
beta strep
b) PYR test (pyrrolidonyl beta naphthylamide test )
(+) result : bright cherry red (beta naphthylamide) upon adding 0.01%
of cinnamaldehyde reagent (p-dimethyl-aminocinnamaldehyde)
(-) result : orange color or no color change
Other PYR test + organisms: Enterococcus, Aerococcus and Gemella
c. Sulfamethoxazole and Trimethoprim (SXT) Test
(+) result resistant (groups A and B) absence of inhibition
Group B ( Streptococcus agalactiae )
- normal flora of the GIT, pharynx, lower GIT and vaginal tract
- nosocomially transmitted by unwashed hands of mother or health care
personnel

- can cause neonatal meningitis (infection of fetuses and infants during

passage through the colonized birth canal and premature rupture of mothers
membranes), sepsis, osteomyelitis
- Resistant to Bacitracin and Bile Esculin Test (-)
- culture: grayish, mucoid with small zone of B-hemolysis
-virulence factor: capsule
Enzymes: hyaluronidase, hemolysis, CAMP factor, neuraminidase,
deoxyribonuclease, protease
Diseases: meningitis, neonatal sepsis, postpartum infection, osteomyelitis,
UTI, endocarditis, pneumonia
Diagnostic tests:
1. CAMP test
- CAMP test test for the identification of Group B streptococci from other bhemolytic strep
Uses b-lysin producing strain of S. aureus or disk impregnated with b-lysin
Perform on ambient gas; (O2 21% with CO2)
Reagent: beta Lysine reagent
(+) result arrow head b-hemolysis near S. aureus growth
2. Hippurate hydrolysis
Reagents: sodium hippurate and ninhydrin
Result: + purple color after adding ninhydrin rgt
(-) no color change

Group C
- Streptococcus equisimus, S. equisimilis, S. dysagalactiae, S. zoopedimicus
- Streptococcus equi causes strangles in horses
Group F and Group G minute streptococci with wide zone of b-hemolysis - S.
anginosus
Group G S. canis found in animals
Laboratory Diagnosis:
Groups C, F and G: Bacitracin (R), SXT (S)
Group D gamma hemolytic
grows at 45 degC and thermoduric; all enterococci isolated from human
hydrolyze PYR
4.1 Enterococcal ( Enterococcus faecalis, E. faecium, E. durans, E.
avium, E. gallinarum)
- normal flora of GUT, GIT, respiratory tract
- grows in 6.5 % NaCl
- resistant to penicillin, vancomycin, cephalosporin; (+) bile
esculin and (+) PYR
E. faecalis requires cyanocobalamin (B12) as growth factor

4.2 Non-Enterococci ( Streptococcus bovis, Streptococcus equinus )

- growth inhibited in 6.5% NaCl
- sensitive to penicillin; PYR test negative
- causes UTI, endocarditis, septicemia
S. bovis mannitol and starch fermenter
Diagnostic tests:
Bile Esculin Hydrolysis Test differentiates Group D from other Streptococci
Presumptive test for Group D, alpha, beta, gamma
1. Growth in bile esculin media esculin hydrolysis tests for the cleavage of
a glycoside
Reagent: esculin + 1-4% bile salt
(+) result black color complex in agar (both enterococci and nonenterococci)
2. 6.5% NaCl (nutrient broth base) test
(+) result turbidity (enterococci), also PYR (+) - Halophilic
(-) result non enterococci = cannot withstand increase concentration of
salt
Leuconostoc, Pediococcus, Globicatella, S. urinalis, Lactococcus (+) bile esculin
and (+) 6.5% NaCl
Isolation of S. bovis from a blood culture may be the first indication that the
patient has an occult tumor in GI tract (GI cancer/colon CA)
Alpha-hemolytic streptococci:
Streptococcus pneumoniae/Diplococcus pneumonia/Pneumococcus
Gram positive cocci in pairs, oval or lancet or bullet shaped diplococcic;
dimple-shaped/donut-shaped colonies
dome-shaped, glistening, wet, mucoid young colonies
coin with a raise rim old colonies
NM, NS, with definite capsule of polysaccharide; Requires 5-10% CO2
Alpha hemolytic under aerobic condition but beta hemolytic under anaerobic
condition
Soluble in bile; Susceptible to optochin
(+) Neufeld Quellung Reaction
Ferment inulin; Susceptible to quinidine
Considered part of normal flora of the upper respiratory tract (25-50%) of
preschool children
One major cause of lobar pneumonia, otitis media, meningitis, peritonitis,
bacteremia, endocarditis, pericarditis
Principal virulence factor: antiphagocytic capsular polysaccharide type 3
strains
Optochin test/Taxo P presumptive ID test for pneumococcus
Bile solubility - susp of pneumococcus is added to bile salt, cloudiness of
broth clears after 3 hours of incubation

Neufeld Quellung antipneumococcal serumis mixed with CSF, sputum with

methylene blue
Mouse virulence
Inulin fermentation
Enzymes: hemolysin, immunoglobulin A protease, neuraminidase and
hyaluronidase
Difference between Streptococci and Pneumococci
Pneumococci
Bile Solubility
Bile Soluble
Inulin Fermentation
ferment inulin
Capsular Swelling
Positive
Quinidine & Optochin
Sensitive

Streptococci
Not soluble
does not ferment
Negative
Resistant

Laboratory Diagnosis
1. Gram stain
2. Culture BHI, TSA with 5% sheeps blood, CAP
3. Susceptibility test Optochin
4. Bile solubility test confirmatory test; differentiate pneumococcus from
viridans
(heavy suspension of pneumococcus is added to bile salt, cloudiness of broth
clears after 3 hrs of incubation
5. Neufeld Quellung Capsular Swelling Test (antipneumococcal serum mixed
with sputum, CSF with methylene blue, examine under OIO = swelling of
capsule
6. Skin test (Francis test)
7. Mouse Virulence Test
8. Inulin Fermentation

Viridans group S. mutans dental caries

do not fall on the criteria for S. pneumonia; can be nonhemolytic; Oropharyngeal
commensals
Glucans and dextrans (extracellular polysaccharides) enhance attachment to host
cell surfaces
Laboratory tests:
1. Bile solubility test
2. Optochin test
3. Growth in 6.5% NaCl

4. Inulin fermentation and PYR

Species: S. mutans, S. salivarius, S. mitis, S. sanguis SBE)
Infections: subacute endocarditis, dental caries/plaque
Gram negative aerobic cocci
Genus: Neisseria, Moraxella, Branhamella, Acinetobacter
Neisseria
General Characteristics:

Non-motile; All are catalase (+) except N. elongata

Aerobic; All species are oxidase (+); Capnophilic

Neisseria meningitidis

These are bean shaped diplococcic; NS, NM, with polysaccharide capsule
Ferments glucose, maltose; Does not ferment lactose , sucrose and fructose
beta lactamase negative; (+) Neufeld Quellung reaction
Specimen: blood and CSF
Leading cause of bacterial meningitis and meningococcemia Waterhouse
Friderichsen Syndrome

Neisseria gonorrhoea

Kidney or bean shaped diplococcic; NM, NS,NE

Requires immediate incubation in CO2 for growth
Not inhibited by vancomycin or lincomycin, colistin and nystatin or
anisomycin
Ferments only glucose; Maltose, lactose, sucrose and fructose (-)

Media:
1. Thayer Martin agar contains colistin, vancomycin or lincomycin, nystatin
2. Modified Thayer Martin media same components with Thayer martin but
with trimetophrim lactate
3. Transgrow media TM with glucose, .2% agar, trimetophrim lactate and
CO2 incorporated in a bottle
4. Martin Lewis media- anisomycin is used instead of nystayin

5. New York City media same antibiotics as Modified TM but amphotericin B

was used as substitute for Nystatin
N.lactimica is a weak nasopharyngeal pathogen that is sometimes mistaken for
N.meningitidis
Veillonellaceae - only anaerobic family
Genus Veillonella, Acidaminococcus, Megasphaera
forms pairs or short chains but smaller than Neisseria
found in the gastrointestinal tracts of animals, but Veillonella are found in
considerable numbers within the oral cavity
mostly non-pathogenic