• C-carbohydrate - serologically active

Streptococcaceae • Detected by antibodies

November 11, 2020 Group A (S. pyogenes) Rhamnose N-acetyl
Marx P. Catalan, RMT, MSMT glucosamine

General Characteristics Group B (S. agalactiae) Rhamnose-glucosamine

• Spherical to ovoid, catalase (-), gram (+) organisms polysaccharide
that tend to occur in pairs or chains when grown in
culture media
• Non-spore former and generally non-motile except
Enclonar, Kimberly / MLS 3A
for rare motile strains of Group D Streptococci •
Generally non-encapsulated except for some strains of Group D (Enterococci &
groups A, B, C & D Streptococci Glycerol teichoic acid
Non-enterococci)
Bergey's Academic Classification (PVEL) containing D-alanine and
• Based on the physiologic divisions on Streptococcus glucose
on: Thermal Requirement
• Obsolete
A Pyogenic
Group A
• Grows neither 45C nor 10C but
Streptococci Characteristics
grows at 11-44C; ave 37C • Colonies are pinpoint
• Transparent to translucent
• Produce pus and mostly beta
hemolytic • Convex or domed entire, circular, shiny
• Surrounded by a wide zone of B-hemolysis
• Ex. S. pyogenes
• Rep: Streptococcus pyogenes
B Viridans • Grows at 45C but not at
Steptococci 10C • Found in normal flora
of the mouth
• Ex. S. salivarius (tongue);
S. mutans (tooth
enamel); S.
sanguis; S. anginosus
Virulence Factors
• Ex. S. mutans - agent of
dental carries (plaque) Cellular components and Extracellular
products • Lipotechoic acid and protein F
C Enterococcus • Grows at both 45C and o Present in the cell wall
group 10C • Ex. S. faecalis (normal o Responsible for the adherence of the bacteria to
fecal flora) the respiratory epithelial cells
• M cells
D Lactic group • Grows at 10C but not at 45C • o Major virulence factor of Group A Streptococci
Ex. S. cremoris; S. lactis (agent which renders the organisms resistant to
of souring of milk/rancid) phagocytosis
• Hyaluronic acid capsule
o Assist the organism in avoiding phagocytosis
Smith and Brown Classification
Hemolysins
• Based on hemolytic patterns on BA
• Streptolysisn O (SLO)
• Alpha hemolytic o Membrane-damaging extracellular toxin
o Partial hemolysis of RBC, accompanied by a
produced by hemolytic streptococci
greenish discoloration o Antigen/immunogenic
o Ex. S. pneumoniae, S. viridans (green
o Triggers production of Anti-Streptolysin O
streptococci) o Oxygen labile
• Beta hemolytic o Causes: subsurface hemolysis on BA
o Complete hemolysis of RBCs indicated by a clear
• Streptolysin S (SLS)
colorless zone of hemolysis surrounding the o Oxygen stable
colonies o Non-antigenic (cannot produce antibodies)
o S. pyogenes, S. agalactiae o Causes surface hemolysis on BA
• Gamma hemolytic
o Non-hemolytic or indifferent Streptococci Enzymes and Toxins
o No hemolysis on RBCs
• Erythrogenic (pyrogenic) toxins
o Ex. S. faecalis o Responsible for the characteristics rash in Scarlet
fever
Lancefield Classification o Strawberry tongue
• Based on the presence of serologically active C • Hyaluronidase
polysaccharide, a group of specific carbohydrate • o Spreading factor
Most commonly used scheme
• Streptokinase
• By Rebecca Lancefield (1933) o Enzyme that dissolves clots
• Not all Streptococci have C-carbohydrate
 Tests for Scarlet Fever phagocytosis
Dick's test • Hemolysins, CAMP factor, neuraminidase, Dnase,
• To determine susceptibility to Scarlet fever • hyaluronidase, protease
Dick's toxin is capable of stimulating a reaction •
Determines previous exposure Biochemical Tests
Hippurate hydrolysis
• Prerequisite: Gram-positive, catalase negative, beta
hemolytic, bacitracin resistant
• This test is used to determine whether a
microorganism, by action of the enzyme hippurate
hydrolase, can hydrolyse sodium hippurate to benzoic
acid and glycine
• Positive result: purple colored product
Enclonar, Kimberly / MLS 3A
Schultz-Charleton test (Blanching Phenomenon)

o Symptoms:
• This is used to diagnose scarlet fever or other diseases
that causes exanthems (rashes on the skin)
• Preformed anti-toxin
• Positive: blanching (neutralization)

Biochemical Tests
Bacitracin (Taxo A)
• Prerequisite: gram positive cocci, catalase negative •
A presumptive test which differentiates group A B
hemolytic Streptococci (+) from other B-hemolytic
Streptococci (-)
• Based on the selective inhibition of the growth of
Group A streptococci by a paper disc containing
0.02- 0.04 units of Bacitracin
• Results: any zone of inhibition regardless of the
diameter is a (+) reaction
• Susceptible: Groups A, C, G
• Resistant: Other B-hemolytic streptococci

PYR Test
• Prerequisite: gram positive cocci, catalase negative,
beta-hemolytic
• Detects the organism's ability to hydrolyze the
substrate L-pyrrolidonyl-beta-napthlamide
• Reagent/color developer: p-dimethyl
aminocinammaldehyde
• Results: bright red end product
• Group A and Enterococci
o Puffy face, "tea or Coca-cola" colored urine
o Hypervolemia secondary to fluid retention
o Hypertension

Group B
Characteristics
• Colonies are large, mucoid, more translucent to
opaque, whitish gray, soft, smooth colonies
surrounded by a smaller zone of beta-
hemolysis • Rep: Streptococcus agalactiae

Virulence Factors
• Capsule made of sialic acid
o Most important virulence factor that prevents
Disease Association • Tolerant in 40% bile
Mode of Transmission • Positive: Dark brown or black color
• Not airborne Enclonar, Kimberly / MLS 3A
• Aerosols or respiratory secretions • Majority appear as diploocci (lancet shaped) • Can be
• Food and milk (epidemics) isolated on sheep BA, and required 5-10% CO2
(capnophilic) showing a wide zone of alpha-hemolysis •
Disease Produced Young colonies (18-24hrs) produce small, round,
• Most common cause of acute pharyngitis: Classic glistening, dome-shaped colonies that are transparent
strep throat with red swollen tonsils and pharynx • with an entire edge
Exudate in tonsils (S. pyogenes) • When colonies become old, autolytic changes result
• Skin infections (impetigo, cellulitis, erysipelas, wound in a collapse of the center of the colony (with raised
infection, pueperal sepsis (sepsis obtained by margins and depressed centers), giving it an
newborn after vaginal delivery), scarlet fever, umbilicate or doughnut appearance (checker or
streptococcal toxic shock syndrome, necrotizing nailhead colonies)
fasciitis (flesh eating)

Sequellae
• Acute Rheumatic Heart fever (Molecular
Mimicry/Type II hypersensitivity)
o Occurs only after pharyngitis
o Symptoms:
o Fever
o Myocarditis
o Joint swelling
• Acute Glomerulonephritis or AGN (immune Complex
Deposition/Type III Hypersensitivity
o Seen after pharyngeal or cutaneous infection
CAMP (Christie, Atkins, and Munch-Peterson) Test •
Differentiate group B B-hemolytic from non-group B B-
hemolytic Streptococci
• The CAMP factor is a diffusible, protein-like compound
produced by Streptococcus agalactiae • A characteristic
"arrowhead" hemolytic pattern results when the
organism is streaked perpendicularly to B-hemolytic S.
aureus
• Can act synergistically with S. aureus hemolysins •
Positive result: enhanced hemolysis is indicated by an
arrow-head shaped zone of B-hemolysis at the juncture
of the two organisms

Disease Association
• Normal flora of the pharynx, gastrointestinal and
genitourinary tract and vagina
• Diseases produced:
o Bovine mastitis
o Skin infection, endocarditis
o Most common: etiologic agent of neonatal
sepsis and meningitis
• FYI: common causes of neonatal meningitidis (S.
agalactiae): Group B streptococci, E. coli & N.
meningitidis

Group D Enterococci vs Non-enterococci •

Prerequisite: gram-positive, catalase negative •
Both are bile-esculin positive
• Non-enterococci is not included in Group D anymore

Bile esculin test (Bile esculin agar)

• Used in selective isolation of D. enterococcus from
other streptococci
• Gram positive other than Group enterococci & Non
enterococci are inhibited by the 40% bile (toxic) in
this medium
 Virulence Factors
Enzymes
• Neuraminidase
• Autolysin
Enterococcus o Facilitates the release of pneumolysin O and
Characteristics other toxic proteins or inflammatory substances
• Natural inhabitants of the intestinal tract of humans from cells
and animals • C-substance
o E. faecalis o Reacts with some components of the immune
o E. faecium response resulting in the activation of some
• Most are non-hemolytic or alpha hemolytic, some are nonspecific host immune response
beta-hemolytic
• Have the ability to grow in extreme conditions: Polysaccharide Capsule
o Presence of bile • Many serologic variations
o 6.5% NaCl • Major virulence factor
o Alkaline pH
Disease Association
Viridans Streptococci • Most common cause of bacterial pneumonia (Lobar) •
Characteristics Second most common cause of bacterial meningitis •
• Normal flora of the upper tract, the female genital Otitis media, purulent sinusitis and occasionally
tract, and the gastrointestinal tract peritonitis
• Referred to as the green streptococci
• Some are beta-hemolytic and non-hemolytic • Biochemical Tests
Fastidious, some requiring increased CO2 for growth • Optochin Sensitivity Test
Classified into 5 groups: • Prerequisite: gram positive, catalase negative, alpha
hemolytic
S. mitis group S. mitis, S. pneumoniae, S. sanguis, S.
• Presumptive test for differentiating S. pneumoniae
oralis
from other alpha-hemolytic streptococci (e.g. the
S. mutans group S. mutans, S. sobrinus viridans streptococci)
• Contains ethylhydrocupreine hydrochloride (Taxo P) •
S. salivarius S. salivarius, S. vestibularis Positive reaction is 14-16mm zone of inhibition using a
group 6mm Optochin disk
• Negative: no zone of inhibition
S. bovis group S. equinus, S. gallolyticus, S. • Equivocal: Any zone of inhibition <14mm is
infantarius, S. alactolyticus questionable for pneumococci; the strain is identified
as a pneumococcus only if it is bile-soluble
S. anginosus S. anginosus, S. constellatus,
group S. intermedius

• Viridans and S. pneumonae does not have lancefield

classification

Streptococcus pneumoniae
Characteristics
• Most virulent member of the S. mitis group of
Viridans Streptococci
• Possess a C substance on their cell wall
o C substance is not same with C polysaccharide •
Has a capsule composed of polysaccharides - mucoid
appearance
• Has an absolute requirement for CHOLINE
o Enzyme that degrades surface structures of host
mucus membranes
• Ig proteases
o Enzymes that facilitate bacterial colonization on
mucosal surfaces by eliminating Ig such as IgA
(mucous membranes), IgG, and IgM
• Pneumolysin O
o An oxygen-sensitive toxin that is cytolytic for
cells
 Enclonar, Kimberly / MLS 3A
Bile Solubility Test
• Use to confirm equivocal results in Taxo P
• S. pneumoniae produce a self-lysing enzyme to inhibit
the growth. The presence of bile salt accelerate this
process
• Add 10parts (10mL) of the broth culture of the
organism to be tested to 1 part (1mL) of 2% Na
deoxycholate (bile) into the test tube
• Negative control is made by adding saline instead of
bile to the culture
o Incubate at 37C for 15min
o Record the result after 15min
• Initial reaction: turbidity to clear
• Positive: clear tube

Neufeld Quellang (Capsular Swelling) Reaction

• A biochemical reaction in which ant capsular
antibodies bind to the capsule of a bacterium,
resulting in the capsule to swell or become more
visible, especially under the microscope
• Reagents: anti-sera containing anticapsular
antibodies. Methylene blue.
• Result: capsular swelling

Respiratory tract - beta hemolysis - s pyogenes

Respiratory tract - optochin negative - viridans