GRAM POSITIVE COCCI

Staphylococcus & Streptococcus

DIANA RYSA S. ASUNCION, RMT

COLLEGE OF MEDICAL LABORATORY SCIENCES
GRAM POSITIVE COCCI

LIST OF GRAM POSITIVE COCCI

CATALASE POSITIVE CATALASE NEGATIVE

Streptococcus spp
Staphylococcus spp
S. pneumoniae
S. aureus
S. mitis
S. epidermidis
S. bovis
S. lugdunensis
S. pyogenes
S. haemolyticus
S. agalactiae
S. Saprophyticus
Viridans streptococci
Micrococcus spp
Enterococcus spp
E. faecium
E. faecalis

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GRAM POSITIVE COCCI

CLASSIFICATION

Mahon: Staphylococcus are members of the family Micrococcacae.

Streptococcus and Enterococcus belongs to Enterococcacae.

Bailey: Staphylococcus are combine with Bacillaceae,

Planococcaceae and Listeriaceae

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INTRODUCTION TO GRAM POSITIVE COCCI

GENERAL CHARACTERISTICS
Micrococcus
▪ Gram positive cocci in tetrads
▪ Under Staphylococacceae
▪ Catalase positive, Oxidase negative
▪ Common normal flora of skin, mucosa and
oropharynx with no known virulence

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INTRODUCTION TO GRAM POSITIVE COCCI

GENERAL CHARACTERISTICS
Staphylococcus spp
➢ “staphle” – bunches of grapes
➢ Seen as round, smooth, raised and glistening on solid media
➢ Gram positive cocci in pairs or in clusters
➢ Young cocci stain strongly as gram positive but stains as
gram negative once old
➢ Catalase positive, Oxidase negative
➢ Carbohydrates slow fermenters, produces lactic acid
➢ Facultative anaerobes, resistant to drying, heat
(50ºC for 30 min)
➢ Common normal flora which can be found on nasopharynx,
skin and other mucosal surfaces

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INTRODUCTION TO GRAM POSITIVE COCCI

GENERAL CHARACTERISTICS
Streptococcus
▪ Gram positive cocci in chains
▪ Catalase negative
▪ Facultative anaerobes
▪ Most common encountered infections to man

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DIFFERENCES OF GRAM POSITIVE AND CATALASE POSITVE COCCI

MICROCOCCUS STAPHYLOCOCCUS
Gram positive cocci in Gram positive cocci in
MICROSCOPY
tetrads clusters
Creamy, medium
White, small colonies CULTURE
colonies
Positive MICRODASE TEST Negative
Sensitive BACITRACIN Resistant
Resistant LYSOSTAPHIN Sensitive

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STAPHYLOCOCCACEAE
Staphylococcus & Micrococcus
STAPHYLOCOCCACEAE

Staphylococcus aureus
▪ NF: Anterior nares, nasopharynx, skin, mucosal
surfaces, perineal area
▪ MOT: Through surgical wound or abrasions, fomites,
air or unwashed contaminated hands

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STAPHYLOCOCCACEAE

Staphylococcus aureus
VIRULENCE FACTOR
✓ Enterotoxin – causes food poisoning, non-invasive pathogen
▪ A-D: pathogenic tissue toxin
▪ E: less significant (<50%)
▪ G-J: commensal tissue toxin
✓ Enterotoxin F/Toxic shock syndrome toxin 1 (TSST-1)
▪ Immunity
▪ Cytokines
✓ Exfoliative toxin
▪ Scalded Skin Syndrome/Ritter Lyell Disease
▪ Suppurative/Pyogenic Infection

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STAPHYLOCOCCACEAE

Staphylococcus aureus
VIRULENCE FACTOR
✓ Enterotoxin – causes food poisoning, non-invasive pathogen
▪ A-D: pathogenic tissue toxin
▪ E: less significant (<50%)
▪ G-J: commensal tissue toxin
✓ Enterotoxin F/Toxic shock syndrome toxin 1 (TSST-1)
▪ Immunity
▪ Cytokines
✓ Exfoliative toxin
▪ Scalded Skin Syndrome/Ritter Lyell Disease
▪ Suppurative/Pyogenic Infection

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STAPHYLOCOCCACEAE

Staphylococcus aureus
VIRULENCE FACTOR
✓ Cytotoxin – based on different hemolysin and leukocidin
▪ Alpha: macrophage and platelets
▪ Beta: sphingomyelinase
▪ Delta: less toxin
▪ Gamma: contributing factor of leukocidins
▪ Leukocidin: can lyse neutrophils which suppress phagocytosis
▪ Panton Valentine Factor

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STAPHYLOCOCCACEAE

Staphylococcus aureus
VIRULENCE FACTOR
✓ Enzymes
▪ Coagulase: two forms (1) clumping factors (2) free coagulase
▪ Staphylocoagulase: traps neutrophil into the clot
▪ Clumping factor: based on the enzyme coagulase that adheres to
fibrinogen and fibrin
▪ Protein A – cell wall component that binds to Fc portion of IgG
molecules that weakens immunity
▪ Staphylokinase – dissolves clot that seals the site of injury

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STAPHYLOCOCCACEAE

Staphylococcus aureus
OTHER FACTORS
Local Infection
➢ Inflammation due to:
▪ Hemolysin – attacks RBC
▪ Leukocidin – attacks WBC
▪ Alpha hemolysin – attacks macrophage and platelets
Systemic Infection
➢ Bacteremia to Septicemia
▪ Hyaluronidase – connective tissue
▪ Peptidase – proteins
▪ Lipase – fats

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STAPHYLOCOCCACEAE

Staphylococcus aureus
CLINICAL MANIFESTATION
▪ Folliculitis – inflammation of follicles
▪ Furuncles – advance causes of folliculitis, causes erythema with
yellow green transparent pus on the skin
▪ Carbuncles – collection of furuncles, most invasive and infectious
▪ Food Poisoning – enterotoxin A, B, C, D
▪ Ritter Lyel Disease/SSS
▪ Toxic Shock Syndrome
▪ Osteomyelitis
▪ Septic Arthritis

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STAPHYLOCOCCACEAE

Staphylococcus epidermidis
✓ NF: skin and mucous membrane
✓ MOT: Implantation of contaminated medical devices
✓ VIRULENCE: Slime or biofilm production
✓ DISEASES AND INFECTION
▪ Nosocomial bacteremia
▪ Postsurgical ocular infections

Staphylococcus lugdunensis and Staphylococcus haemolyticus

➢ Almost similar with Staph. epidermidis with fewer numbers
and uncertain virulence factor
➢ Mistaken as coagulase positive due to the presence of
clumping factor

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STAPHYLOCOCCACEAE

Staphylococcus saprophyticus
✓ NF: Skin and mucosa of genitourinary tract
✓ MOT: Sexually acquired infection
✓ DISEASE AND INFECTION: UTI for sexually active females
OTHER STRAINS
Methicillin Resistant Staphylococci
➢ Penicillin resistant strains (nafcillin or oxacillin)
➢ CHROM agar MRSA
➢ mec A: Gold standard for MRSA detection

Vancomycin Resistant Staphylococci

➢ Drug of choice for serious staphylococcal infections
➢ Vancomycin resistant strains

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STAPHYLOCOCCACEAE

LABORATORY DIAGNOSIS

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STAPHYLOCOCCACEAE

MICROSCOPY METHOD
Staphylococcus
➢ Spherical, gram positive cocci in clusters
➢ Known as “grape like bacteria”
Micrococcus
➢ Spherical, gram positive cocci in pairs, tetrads and irregular
clusters which resembles Staphylococcus

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STAPHYLOCOCCACEAE

CULTURE METHOD
5% Sheep blood agar (SBA)
➢ Incubated at 35C in CO2 or ambient air for 24 hours
➢ Round, smooth, white, creamy colonies
▪ Staph aureus: rarely exhibits yellow pigmentation
▪ Staph epidermidis: small to medium sized, gray to white colonies,
non-hemolytic
▪ Staph saprophyticus: larger colonies
▪ Staph haemolyticus: medium colonies with moderate or weak
hemolysis
▪ Staph lugdunensis: often hemolytic, medium colonies

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STAPHYLOCOCCACEAE

CULTURE METHOD
Mannitol Salt Agar (MSA)
➢ Incubated at 35C for at least 48-72 hours
➢ Selective media used to isolate staphylococci
➢ Contains 7.5% salt, mannitol as sugar source and
phenol red as ph indicator
➢ Staph. aureus: yellow halo colonies

Other Culture Media

▪ Colistin Nalidixic Acid (CAN)
▪ Phenylethyl Alcohol (PEA)
▪ Trypticase Soy Agar (TSA)
STAPHYLOCOCCACEAE

RAPID METHODS
Polymerase Chain Reaction
➢ Molecular assays
➢ Identification of MRSA and MSSA
Qualitative Nucleic Acid Hybridization Assay
➢ Targets ribosomal RNA sequences
➢ Detects the presence of staphylococci in blood cultures

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STAPHYLOCOCCACEAE

SENSITIVITY METHODS
Bacitracin
➢ SUS: Micrococcus, Aerococcus
➢ RES: Staphylococcus, Enterococcus
Novobiocin
➢ SUS: Staph epidermidis, Staph lugdunensis
➢ RES: Staph saprophyticus
Lysostaphin
➢ SUS: Staphylococcus
➢ RES: Micrococcus, Aerococcus, Enterococcuss
Furazolidone
➢ SUS: Staphylococcus, Aerococcus, Enterococcus
➢ RES: Micrococcus

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STAPHYLOCOCCACEAE

BIOCHEMICAL TESTS
Catalase
➢ PRIN: mediates the breakdown of
hydrogen peroxide into oxygen and water
within 10 seconds
➢ Separates Staphylococci from
Streptococci
➢ RGT: 3% Hydrogen peroxide
➢ POS: Effervescence formation (Staph)

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STAPHYLOCOCCACEAE

BIOCHEMICAL TESTS
Coagulase
➢ PRIN: binding of bacterial cells wall to fibrinogen
➢ RGT: rabbit’s plasma
➢ Distilled water/NSS: for gram negative with auto coagulation due to
the presence of other organism or old sample
➢ Two types
▪ Slide Method: detects bound coagulase, clumping factors
▪ Tube Method: detects free coagulase, confirmatory
➢ POS: agglutination (Staph aureus)

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STAPHYLOCOCCACEAE

BIOCHEMICAL TESTS
Modified Oxidase Test
➢ PRIN: catalyzes the oxidation of cytochrome c
➢ Differentiates Staphylococcus from Micrococcus
➢ RGT: 6% tetramethyl-p-phenylenediamine
dihydrochloride in dimethyl sulfoxide
➢ POS: blue to purple color (Microccocus)

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Have a break, but don’t break your heart.
STREPTOCOCCACEAE
Streptococcus and Enterococcus
STREPTOCOCCACAEA

CLASSIFICATION OF STREPTOCOCCUS
Smith’s and Brown’s Classification
➢ Also known as JH Brown
➢ Uses blood agar plate
➢ The ability of organism to lyse blood
TYPES OF HEMOLYSIS
▪ Alpha hemolysis: ability to produce H202 that lyses RBC
and releases hemoglobin
▪ Beta hemolysis: ability to produce hemolysis
✓Streptolysin O
❖ O2 labile, antigenic and subsurface
✓Streptolysin S
❖ O2 stable, non –antigenic and surface hemolysin
▪ Non-hemolytic

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STREPTOCOCCACAEA

CLASSIFICATION OF STREPTOCOCCUS
Rebecca Lancefield Classification
➢ C polysaccharide – carbohydrate that is attached to murein layer
Amino sugars present
▪ A: Rhamnose-N-acetyl glucosamine
▪ B: Rhamnose glucosamine
▪ C: Rhamnose-N-acetylgalactoseamine
▪ D: Glycerol Teichoic Acid
▪ F: Glycopyranosyl-N-acetyl-galactosamine

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STREPTOCOCCACAEA

CLASSIFICATION OF STREPTOCOCCUS
Sherman Classification
➢ Academic/Bergeys classification
➢ Based from hemolytic pattern, carbohydrate antigen, phenotype testing
(pH, salt tolerance)
▪ Pyogenic Group
✓Pus production, Beta hemolytic, A to G, Grows at 37C
▪ Viridans Group
✓Grows at 37C and 45C
▪ Lactic Group
✓Milk industry, Grows at 10C and 37C
▪ Enterococcus
✓Salt tolerant and survives in increased pH, Grows on 10C, 37C, 45C

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STREPTOCOCCACAEA

Group Bacteria Hemolytic Pattern

A S. pyogenes Beta
B S. agalactiae Beta
S. equisimilis
S. equi
C Beta
S. zoopedemicus
S. dysagalactiae

E. faecalis
E. faecium
Beta
E. durans
D Alpha
E. avium
Gamma
S. bovis
S. equinus

(Viridans group)
S. mitis (mitior)
S. salivarius
S. uberis
Alpha
S. constellatus
S. intermedius
S. mutans
S. sanguis
---- S. pneumoniae Alpha

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STREPTOCOCCACAEA

ALPHA HEMOLYTIC
Streptococcus pneumoniae
▪ Found in nasopharynx of man
▪ Alpha hemolytic strep, needs CO2 for incubation
▪ No C polysaccharide but contains substances that triggers
chemical reaction with protein
▪ C reactive protein
▪ Capsules are detected using:
✓Neufeld Test
✓Quelling Reaction
✓Capsular Swelling

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STREPTOCOCCACAEA

ALPHA HEMOLYTIC
Streptococcus pneumoniae
▪ VIRULENCE
✓Capsule
✓Hemolysin
✓IgA protease
✓Hyaluronidase
✓Neuramidase
▪ DISEASES AND INFECTION
✓Sinusitis
✓Otitis media (ear infection)
✓Bacteremia
✓Lobar pneumonia
✓Meningitis

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STREPTOCOCCACAEA

ALPHA HEMOLYTIC
Viridans streptococci
▪ NF: Oral cavity, GI tract, Female genital tract
▪ Considered as either alpha, beta, non-hemolytic organism
▪ MOT: Dental manipulations
▪ Five groups
✓ S. mitis
✓ S. Mutans
✓ S. Salivorius
✓ S. bovis
✓ S. anginous

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STREPTOCOCCACAEA

ALPHA HEMOLYTIC
Viridans streptococci
▪ VIRULENCE
✓ Extracellular complex polysaccharide
✓ Cytotoxin
✓ Dextro
✓ Adhesins
▪ DISEASE AND INFECTION
✓ Subacute endocarditis in patients with
damaged heart valves
✓ Infections to immunocompromised patients due
to contamination
✓ Meningitis

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STREPTOCOCCACAEA

BETA HEMOLYTIC
Streptococcus pyogenes (Group A)
▪ Inhabits skin and URT which is considered as clinically significant
▪ Beta hemolytic strep
▪ MOT: Person to person contact with contaminated secretions
▪ VIRUENCE
✓ Protein F/Fibronectin – mediated epithelial cell attachment
✓ M protein – coded by emm gene attached to murein
✓ Toxins – for invasion
❖ Streptococcal Pyodermal Exotoxin: SpE A, SpE B, SpE C, SpE F
✓ Hemolysin
❖ Streptolysin O (SLO), Streptolysin S (SLS)
✓ Enzymes
❖ Streptodornase, Streptokinase, Hyaluronidase, DNase

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STREPTOCOCCACAEA

BETA HEMOLYTIC
Streptococcus pyogenes (Group A)
▪ DISEASE AND INFECTION
✓ Acute pharyngitis
✓ Tonsillitis
✓ Necrotizing fasciitis
✓ Scarlet fever
✓ Streptococcal toxic shock syndrome
✓ Sequelae infection
❖ Rheumatic Heart Disease
❖ Acute Rheumatic Fever
❖ Acute Glomerulonephritis

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STREPTOCOCCACAEA

BETA HEMOLYTIC
Streptococcus agalactiae (Group B)
▪ NF: Female genital tract, lower GI
▪ Beta hemolytic strep
▪ MOT: Parenteral during delivery, nosocomial transmission
▪ VIRULENCE
✓ Sialic Acid Capsule – interferes with phagocytic activity and
complement cascade activation
✓ CAMP factor
✓ Hemolysin
✓ Neuraminidase
✓ DNase
✓ Hyaluronidase
✓ Protease

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STREPTOCOCCACAEA

BETA HEMOLYTIC
Streptococcus agalactiae (Group B)
▪ DISEASE AND INFECTION
✓ Neonatal Infection
➢ 35th to 37th GBS testing
❖ Pneumonia
❖ Meningitis
❖ Sepsis
✓ Adult Infection
➢ Skin and soft tissue
➢ Pneumonia
➢ Sepsis arthritis
➢ UTI, Bacteremia
➢ Endocarditis

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STREPTOCOCCACAEA

NON-HEMOLYTIC
Enterococcus
▪ Found in soil and water
▪ Considered as either alpha, beta or non-hemolytic strep
▪ MOT: Person to person, direct or by contaminated medical equipment
▪ VIRULENCE
✓ Cytolysin
✓ Multidrug resistant pathogen
▪ DISEASE AND INFECTION
✓ UTI
✓ Bacteremia
✓ Endocarditis

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STREPTOCOCCACAEA

LABORATORY DIAGNOSIS
STREPTOCOCCACAEA

MICROSCOPIC METHOD
Streptococcus
▪ Gram positive cocci in pairs or in chains
▪ Round, oval shaped, some are elongated
▪ Becomes gram negative if the cultures are old and
patients have taken antibiotics

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STREPTOCOCCACAEA

CULTURE METHOD
5% Sheep blood agar
➢ Differential media based on the hemolytic pattern
➢ Incubated with 5-10% carbon dioxide
▪ Alpha hemolytic: partial hemolysis
▪ Beta hemolytic: complete hemolysis
▪ Non hemolytic: no hemolysis

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STREPTOCOCCACAEA

CULTURE METHOD
Other Selective Medium for Streptococci
▪ 5%SBA with STX – suppresses the growth of NF
▪ Todd Hewitt broth with antimicrobials – suppresses the
growth of vaginal flora

SENSITIVITY METHOD OTHER TESTS

Optochin (Taxo P)
➢ SUS: Strep pneumoniae
➢ RES: Strep mitis
Bacitracin (Taxo A)
➢ SUS: Strep pyogenes
➢ RES: Strep agalactiae
Serodiagnosis
▪ Anti-streptolysin O (ASO)
▪ Anti-DNase B
▪ Anti-streptokinase

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STREPTOCOCCACAEA

CULTURE METHOD

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STREPTOCOCCACAEA

BIOCHEMICAL METHOD
CAMP Test
▪ PRIN: CAMP factor acts synergistically with beta lysin of
Staph aureus causing enhanced hemolysis
▪ Differentiates Strep agalactiae to other beta hemolytic
streptococci
▪ RGT: Staph aureus on SBA
▪ POS: enhanced hemolysis, arrow head

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STREPTOCOCCACAEA

BIOCHEMICAL METHOD
Hippurate Test
▪ PRIN: production of hydrolysis by the
deamination of glycine forming ninhydrin
where ninhydrin oxidizes due to the
presence of free amino acids to form
hippuric acid
▪ RGT: hippurate disk
▪ POS: deep purple color

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STREPTOCOCCACAEA

BIOCHEMICAL METHOD
Bile Solubility Test
➢ PRIN: lysis of pneumococcal colonies due to
the presence of bile or bile salt
➢ Differentiates Strep pneumoniae from other
alpha hemolytic streptococci
➢ RGT: 10% sodium desoxycholate (slide)
2% sodium desoxycholate (tube)
➢ POS: disintegrated colonies (slide)
clearing of turbidity (tube)

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STREPTOCOCCACAEA

BIOCHEMICAL METHOD
Bile Esculin Test
➢ PRIN: Potential to growth due in the presence
of 40% bile and the ability to hydrolyze
esculin which turns the indicator ferric
ammonium citrate
➢ RGT: 40% bile, esculin
➢ POS: black color (Enterococcus faecalis)

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STREPTOCOCCACAEA

BIOCHEMICAL METHOD
Salt Tolerance Test
➢ PRIN: ability of organism to grow in high
salt concentration
➢ Differentiates enterococci to non-
enterococci
➢ RGT: 6.5% NaCl, Todd Hewitt broth
➢ POS: visible turbidity with or without color
change from purple to yellow
(Enterococcus faecalis)

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STREPTOCOCCACAEA

BIOCHEMICAL METHOD
PYR Test
▪ PRIN: ability of L-pyrroglutamylaminopeptidase to hydrolize L-
pyrrolidonyl-b-napthylamide to produce beta napthylamine
▪ RGT: N, N-dimethylaminocinnamaldehyde
▪ POS: bright red color within 5 minutes

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 LABORATORY SEQUENCE
GRAM STAIN

Gram Positive Gram Negative

Catalase Test
Coagulase Hemolysis

S. aureus Alpha Beta Non

Bacitracin susceptibility
S R Optochin disk Bacitracin
Micrococcus spp. CoNS S R S R
Oxidase (+) blue color S. pneumoniae S. pyogenes
Novobiocin susceptibility Bile esculin CAMP Bile esculin
S R Viridans streptococci
CoNS S. saprophyticus S. agalactiae Non hemolytic
Hippurate hydrolysis (+)
streptococci

PYR
Enterococcus spp. Group D Streptococci
THANK YOU ☺
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