Streptococcus,

TYPES OF HEMOLYSIS
Types of Hemolysis

Enterococcus, and Other Hemolysis

Alpha (α)
Description
Partial lysis of RBCs

Catalase-Negative, Gram- Beta (β)

around colony
Complete lysis of RBCs

Positive Cocci around colony

Clear area around colony
Nonhemolytic No lysis of RBCs around
colony
Definition of Terms No change in agar
Pathogenicity Alpha-prime (α′) Small area of intact RBCs
or wide zone around colony
surrounded by a wider
General Characteristics:
zone of complete
Streptococcus and Enterococcus hemolysis
 belong to the family Streptococcaceae RBC, Red blood cell.
 catalase-negative, gram-positive cocci that are
usually arranged in pairs or chains
 facultative anaerobes, should be considered
aerotolerant anaerobes
 Some species are capnophilic

Cell Wall Structure Clinically Significant Streptococci and

• have a group or common C carbohydrate
Streptococcus-like Organisms
(polysaccharide)
Classification of Streptococcus and Enterococcus
• Rebecca Lancefield
Species Lance Hemo Common Disease
• Lancefield was able to divide the streptococci into field lysis Terms Association
serologic groups, designated by letters. Group Types (s)
Antig
en
S. A β Group A Rheumatic
pyogen streptococ fever,
es ci scarlet
fever,
pharyngitis,
glomerulon
ephritis,
pyogenic
infections
 S. B β Group B Neonatal  Are adhesion molecules that mediate
agalacti streptococ sepsis, attachment to host epithelial cells.
ae ci meningitis, • Hyaluronic acid capsule
puerperal
 Weakly immunogenic.
fever,
pyogenic  The capsule prevents opsonized phagocytosis
infections by neutrophils or macrophages.
S. C β Group C Endocarditi  The capsule also allows the bacterium to mask
dysgala streptococ s, UTIs, its antigens and remain unrecognized by its
ctiae, S. ci pyogenic host.
equi infections • Streptolysin O
S. bovis D α, Nonentero Endocarditi
 responsible for hemolysis on SBA plates
group none coccus s, UTIs,
member pyogenic incubated anaerobically.
of viridans infections  Oxygen labile.
streptococ  lyses leukocytes, platelets, and other cells as
ci well as RBCs.
E. D α, β, Enterococ UTIs,  SLO is highly immunogenic
faecalis none cus pyogenic
• Streptolysin S
, E. infections
faecium  Oxygen stable
S. — α Pneumoco Pneumonia,
 lyses leukocytes, and is nonimmunogenic.
pneum ccus meningitis,
oniae pyogenic • Hyaluronidase, or spreading factor
infections  an enzyme that solubilizes the ground
Anginos A, C, β, α, Viridans Pyogenic substance of mammalian connective tissues
us F, G, none streptococ infections, (hyaluronic acid).
group, N, or ci endocarditi • streptococcal pyrogenic exotoxins,
mutans — s, dental
 erythrogenic toxins
group, caries,
mitis abscesses in  causes a red spreading rash, referred to as
group, various scarlet fever,
salivari tissues
us CLINICAL INFECTIONS
group 1. Bacterial Pharyngitis
• pharyngitis and tonsillitis
Clinically Significant Streptococci and • “Strep throat”
Streptococcus-like Organisms
Streptococcus pyogenes
• Lancefield group A
• M protein is attached to the peptidoglycan of the
cell wall and extends to the cell surface.
2. Pyodermal Infections
• Colonizes the throat and skin on humans, making
• Impetigo
these sites the primary sources of transmission
VIRULENCE FACTORS  a localized skin disease, begins as small
• M protein vesicles that progress to weeping lesions.
 encoded by the genes emm.  Usually seen in young children (2 to 5 years)
and affect exposed areas of the skin.
 causes the streptococcal cell to resist
• Erysipelas
phagocytosis and plays a role in adherence of
the bacterial cell to mucosal cells.
• Lipoteichoic acid and protein F
  a rare infection of the skin and 4. Streptococcal Toxic Shock Syndrome.
subcutaneous tissues observed frequently • a condition in which the entire organ system
in elderly patients. collapses, leading to death
 Characterized by an acute spreading skin • The initial streptococcal infection is often severe
lesion that is intensely erythematous with a (e.g., pharyngitis, peritonitis, cellulitis, wound
plainly demarcated but irregular edge. infections), and the symptoms that develop are
similar to symptoms of staphylococcal TSS.
5. Poststreptococcal Sequelae.
• rheumatic fever and acute glomerulonephritis.

LABORATORY DIAGNOSIS
1. SPECIMEN COLLECTION
• Cellulitis • The tongue should be depressed and the swab
 can develop following deeper invasion by rubbed over the posterior pharynx and each
streptococci. tonsillar area
 The infection can be serious or life- • If exudate is present, it should also be touched
threatening with bacteremia or sepsis. with the swab.
• Scarlet fever • Care should be taken to avoid the tongue and
 characterized by a diffuse red rash that uvula.
appear s on the upper chest and spreads to 2. GRAM STAINING
the trunk and extremities. • Gram stain reveals gram-positive cocci with
 The rash disappears over the next 5 to 7 some short chains
days and is followed by desquamation. 3. CULTURE
• SBA plate- small, transparent, and smooth with
a well-defined area of β-hemolysis
4. BIOCHEMICAL TESTS
• bacitracin susceptibility or pyrrolidonyl α-
naphthylamide (PYR) hydrolysis
• hippurate hydrolysis
• CAMP test
• bile esculin test
• growth in 6.5% sodium chloride (NaCl) broth.

Streptococcus agalactiae
3. Necrotizing Fasciitis. • group B–specific antigen, an acid-stable
• an invasive infection characterized by rapidly polysaccharide located in the cell wall.
progressing inflammation and necrosis of the
skin, subcutaneous fat, and fascia. VIRULENCE FACTORS:
• Although uncommon, NF is a lifethreatening 1. Capsule
infection 2. Hemolysin
• “flesh-eating disease” 3. CAMP factor
No evidence exists that any of these
products plays a role in the virulence of this
organism.
4. Neuraminidase • large-colony–forming isolates - S. dysgalactiae
5. Dnase subsp. Equisimilis
6. Hyaluronidase • small-colony–forming β-hemolytic - S. anginosus
7. protease • The spectrum of infections resembles S. pyogenes
and includes upper respiratory tract infections, skin
CLINICAL INFECTIONS infections, soft tissue infections, and invasive
1. Mastitis in cattle infections such as NF.
2. Neonatal GBS disease • S. equi subsp. zooepidemicus - primarily an animal
• early-onset infection (<7 days old) – manifests pathogen rarely isolated from humans.
as pneumonia and sepsis Streptococcus pneumoniae
• late-onset infection (at least 7 days old to about
3 months old). – manifests as meningitis and Streptococcus pneumoniae
sepsis • Also known as pneumococcus
• a member of the S. mitis group, but it is much more
virulent than other members of the group
• The cell wall contains an antigen, referred to as C
substance, which is similar to the C carbohydrate of
the various Lancefield groups

VIRULENCE FACTORS
• Capsular polysaccharide
• hemolysin,
LABORATORY DIAGNOSIS
• immunoglobulin A protease - destroys IgA in the
SPECIMEN COLLECTION:
respiratory tract assiting in colonization
• collecting vaginal and rectal material with swabs
• Neuraminidase - enhance adherence to mucous
between 35 and 37 weeks of gestation
membrane surfaces by cleaving sialic acid exposing
GRAM STAIN: molecules enhancing adhesion
• gram-positive cocci that form short chains in clinical • Hyaluronidase
specimens and longer chains in culture CLINICAL INFECTIONS: Pneumonia; Sinusitis & Otitis
CULTURE: media; Bactermia; Septicemia; Meningitis; Endocarditis;
• SBA- grayish white mucoid colonies surrounded by a septic arthritis; soft tissue infections
small zone of β-hemolysis
LABORATORY DIAGNOSIS
BIOCHEMICAL TESTS: GRAM STAIN:
• Hippurate hydrolysis • grampositive cocci in pairs (diplococci).
• CAMP tests • The ends of the cells are slightly pointed, giving
them an oval or lancet shape

CULTURE:
• Brain-heart infusion agar
• Trypticase soy agar with 5% sheep RBCs
• Chocolate agar
• SBA

Colonial Morphology:
Groups C and G Streptococci • round, glistening, wet, mucoid, domeshaped
appearance, a-hemolytic on SBA
BIOCHEMICAL TESTS:
• Optochin susceptibility
• Bile solubility
appearance of a “ coin with a raised rim”
Viridans Streptococci
• constituents of the normal microbiota of the upper
respiratory tract, the female genital tract, and the
gastrointestinal tract.
• viridans means “green,” referring to the α-
hemolysis many species exhibit.
• oropharyngeal commensals that are regarded as
opportunistic pathogens
FIVE GROUPS:
1. S. mitis group (including S. mitis, S. pneumoniae, S.
sanguis, S. oralis)
2. S. mutans group (including S. mutans and S.
sobrinus)
3. S. salivarius group (including S. salivarius and S.
vestibularis);
4. S. bovis group (including S. equinus, S. gallolyticus,
S. infantarius, and S. alactolyticus)
5. S. anginosus group (including S. anginosus, S.
constellatus, and S. intermedius).
CLINICAL INFECTIONS:
1. Subacute bacterial endocarditis
• Viridans streptococci are the most common
cause
• a condition associated with a transient
bacteremia.
2. oral infections such as gingivitis and dental caries
(cavities)
LABORATORY DIAGNOSIS
GRAM STAIN: • typical Streptococcus characteristics on
Gram stain. COLONIAL MORPHOLOGY • small and are
surrounded by a zone of α-hemolysis; some isolates are
β-hemolytic or nonhemolytic BIOCHEMICAL TESTS: •
Voges-Proskauer (VP) • β-D-glucuronidase activity •
hippurate hydrolysis • PYR • Leucine aminopeptidase
(LAP) LABORATORY DIAGNOSIS Enterococcus
•previously classified as group D streptococci •This
group consists of gram-positive cocci that are natural
inhabitants of the intestinal tracts of humans and
animals. •Most enterococci are nonhemolytic or α-
hemolytic, although some strains show β-hemolysis.
•Enterococci sometimes exhibit a pseudocatalase
reaction—weak bubbling in the catalase test VIRULENCE
FACTORS 1. Extracellular surface adhesin proteins 2.
extracellular serine protease 3. Gelatinase 4. Cytolysin
Clinically Significant Streptococci and Streptococcus-like
Organisms play a role in the colonization of the species
and adherence to heart valves and renal epithelial cells.
Enterococcus CLINICAL INFECTIONS frequent causes of
nosocomial infections •UTI •Bacteremia •Endocarditis-
seen in elderly patients with prosthetic valves or
valvular heart disease Clinically Significant Streptococci
and Streptococcus-like Organisms Enterococci 1.
SPECIMEN COLLECTION • Standard procedures for
collection and transport of blood, urine, or wound
specimens should be followed. • The specimens should
be cultured as soon as possible with minimum delay 2.
GRAM STAINING • Gram stain reveals gram-positive
cocci with some short chains LABORATORY DIAGNOSIS
3. CULTURE • Trypticase soy or brain-heart infusion agar
supplemented with 5% sheep blood • bile esculin azide
• colistin– nalidixic acid • phenylethyl alcohol •
chromogenic substrates • cephalexin-aztreonam-
arabinose agar Enterococci BIOCHEMICAL TESTS: •
Enterococcus spp. are identified based on their: 1.
ability to produce acid in carbohydrate broth 2. ability
to hydrolyze arginine 3. tolerance of 0.04% tellurite 4.
utilization of pyruvate 5. ability to produce acid from
methylα-D-glucopyranoside 6. growth around 100-µg
efrotomycin acid disk 7. motility. LABORATORY
DIAGNOSIS 1. Abiotrophia and Granulicatella • formerly
known as the nutritionally variant streptococci • These
bacteria grow as satellite colonies around other bacteria
and require sulfhydryl compounds for growth. • part of
the human oral and gastrointestinal microbiota •
significant cause of bacteremia, endocarditis, and otitis
media 2. Aerococcus • a common airborne organism. •
It is a widespread, opportunistic pathogen associated
with bacteremia, endocarditis, and UTI in
immunocompromised patients. • resemble viridans
streptococci on culture but are microscopically similar
to staphylococci in that they occur as tetrads or clusters
•Aerococcus urinae is a cause of UTI, endocarditis,
lymphadenitis, and peritonitis. Streptococcus-like
Organisms 3. Gemella • similar in colony morphology
and habitat to viridans streptococci • α-hemolysis or are
nonhemolytic. • The bacteria easily decolorize on Gram
staining and often appear as gram-negative cocci in
pairs, tetrads, clusters, or short chains. • Isolated from
cases of endocarditis, wounds, and abscesses. • The
most significant species is Gemella haemolysans. 4.
Lactococcus • gram-positive cocci that occur singly, in
pairs, or in chains and are physiologically similar to
enterococci. • α-hemolysis or are nonhemolytic. •
previously classified as group N streptococci. • isolated
from patients with UTI and Endocarditis Streptococcus-
like Organisms 5. Leuconostoc • catalase-negative,
grampositive microorganisms with irregular coccoid
morphology • Some species cross-react with the
Lancefield group D antiserum. • resistant to
vancomycin. • In nature, they are frequently found on
plant surfaces and vegetables, and in milk products. •
They are recognized as opportunistic pathogens • These
microorganisms have been isolated from cases of
meningitis, bacteremia, UTIs, and pulmonary infections.
• Species associated with infection include: •
Leuconostoc citreum • Leuconostoc cremoris •
Leuconostoc dextranicum • Leuconostoc lactis •
Leuconostoc mesenteroides • Leuconostoc
pseudomesenteroides. Streptococcus-like Organisms 6.
Pediococcus • facultatively anaerobic, gram-positive
cocci (arranged in pairs, tetrads, and clusters) • grows at
45° C. resistant to vancomycin. • associated with
patients who have underlying gastrointestinal
abnormalities or who have previously undergone
abdominal surgery • The following species have been
associated with infection: • Pediococcus acidilactici •
Pediococcus damnosus • Pediococcus dextrinicus •
Pediococcus parvulus • Pediococcus pentasaceus.
Classification Schemes 1. Hemolytic pattern on SBA 2.
Physiologic characteristics 3. Serologic grouping or
typing of C carbohydrate (Lancefield classification),
capsular polysaccharide, or surface protein, such as the
M protein of S. pyogenes 4. Biochemical characteristics
LABORATORY DIAGNOSIS Biochemical tests used for
identification of Streptococci BACITRACIN
SUSCEPTIBILITY TEST PURPOSE • To differentiate
Streptococcus pyogenes from other β-hemolytic
streptococci PRINCIPLE • Group A streptococci are
susceptible to low levels (0.04 units) of bacitracin,
whereas other groups of β-hemolytic streptococci are
resistant. SPECIMEN • Isolated colonies of test organism
on sheep blood agar MEDIA • 5% sheep blood agar
plate REAGENT •Bacitracin disk, 0.04 units PROCEDURE
1. Streak surface of agar plate to obtain isolated
colonies. 2. 2. Aseptically place bacitracin disk onto
inoculated surface. Press down gently on the disk to
ensure complete contact with the agar surface. 3.
Incubate the plate at 35° C for 18 to 24 hours in a CO2
incubator. INTERPRETATION Susceptible = Any zone of
inhibition around the bacitracin disk Resistant =
Uniform lawn of growth up to the edge of the disk
Biochemical tests used for identification of Streptococci
CAMP TEST PURPOSE • To differentiate Streptococcus
agalactiae from other β-hemolytic streptococci
PRINCIPLE • Streptococcus agalactiae produces CAMP
factor, which enhances the lysis of sheep red blood cells
by staphylococcal β-lysin. A positive reaction can be
observed in 5 to 6 hours with incubation in CO2 (18
hours with incubation in ambient air). SPECIMEN 1.
Isolated colonies of test organism on sheep blood agar
2. 2. β-Lysin–producing S. aureus on sheep blood agar
MEDIUM • sheep blood agar plate PROCEDURE 1.
Inoculate S. aureus along a line down the center of the
agar plate. 2. 2. Inoculate the streptococcal isolates
along a thin line about 2 cm long and perpendicular to,
but not touching, the S. aureus streak. 3. Incubate plate
at 35° C for 18 hours. INTERPRETATION • Positive result
= Arrowhead-shaped area of enhanced hemolysis •
Negative result = No enhanced hemolysis • CAMP
inhibition reaction (reverse CAMPpositive) = Inhibition
of hemolysis by S. aureus where the two streaks
approach each other (Arcanobacterium haemolyticum)
Biochemical tests used for identification of Streptococci
Hippurate Hydrolysis Test PURPOSE • To differentiate
Streptococcus agalactiae from other β-hemolytic
streptococci PRINCIPLE • The enzyme hippuricase
hydrolyzes hippuric acid to form sodium benzoate and
glycine. Subsequent addition of ninhydrin results in the
release of ammonia from the oxidative deamination of
the α amino group in glycine as well as the reduced
form of ninhydrin, hydrindantin. The ammonia reacts
with residual ninhydrin and hydrindantin to produce a
purple-colored complex. SPECIMEN • Sodium hippurate
• Ninhydrin Reagent PROCEDURE 1. Inoculate the
solution of sodium hippurate heavily with colonies 18 to
24 hours old until a milky suspension is obtained. 2.
Incubate tube for 2 hours at 35° C. 3. Add 0.2 mL of
ninhydrin reagent. 4. Mix and incubate for 10 to 15
minutes at 35° C. INTERPRETATION • Positive result =
Deep purple color—indicates hippurate hydrolysis •
Negative result = No color change or very slight purple
color Biochemical tests used for identification of
Streptococci Pyrrolidonyl-α-Naphthylamide Hydrolysis
PURPOSE • To differentiate gram-positive cocci that will
hydrolyze the substrate L-pyrrolidonyl α-nap hthylamide
(PYR) from those that are PYR-ne gative PRINCIPLE •
PYR-impregnated disks serve as the substrate to
produce α-naphthylamine, which is detected in the
presence of D-dimethylaminocinnamaldehyde (DMCA)
by the production of a red color. PROCEDURE 1. Lightly
moisten a PYR-impregnated disk with sterile water. 2.
Using a sterile loop, rub one or more isolated colonies
onto the surface of the disk. 3. NOTE: Incubation time
and temperature vary slightly by manufacturer.
Incubate disk as indicated in the manufacturer’s
instructions, generally 2 to 15 minutes. 4. Add a drop of
color developer and observer for a red color on the disk
within 5 minutes. INTERPRETATION • Positive result =
Red color • Negative result = Colorless Biochemical tests
used for identification of Streptococci Bile Esculin
Hydrolysis PURPOSE • To differentiate group D
streptococci and enterococci from other catalase-
negative, gram-positive cocci PRINCIPLE • Group D
streptococci and enterococci grow in the presence of
bile and also hydrolyze esculin to esculetin and glucose.
Esculetin diffuses into the agar and combines with ferric
citrate in the medium to produce a black complex.
MEDIA • Bile Esculin Agar PROCEDURE 1. Pick one or
two isolated colonies from the sheep blood agar plate
and inoculate to bile esculin agar medium. A single plate
can be divided into several pie-shaped section s for
inoculation of multiple test organisms. 2. Incubate plate
or slant at 35° C for 18 to 24 hours. A positive result is
often seen within 4 hours. A negative result should be
incubated for an additional 24-hour period.
INTERPRETATION Positive result = Blackening of the
agar Negative result = No blackening of the agar.
**NOTE: Growth alone does not constitute a positive
result. Biochemical tests used for identification of
Streptococci Leucine Aminopeptidase PRINCIPLE: •The
substrate, leucine-β-naphthylamide, is hydrolyzed to β-
naphthylamine. After the additio n of
paradimethylaminocinnamaldehyde reagent, a red color
develops. LAP POSITIVE Streptococcus, Enterococcus
Pediococcus spp. LAP NEGATIVE Aerococcus
Leuconostoc spp. Biochemical tests used for
identification of Streptococci Voges-Proskauer Test.
PURPOSE •used to distinguish the small-colony–forming
β-hemolytic anginosus group containing groups A or C
antigens from large-colony–forming pyog enic strains
with the same antigens. POSITIVE REACTION: Red or
pink color detects the action of β-D-glucuronidase, an
enzyme found in isolates of largecolony– forming β-
hemolytic groups C and G streptococci but not in the
smallcolony–forming β-hemolytic anginosus group. β-D-
Glucuronidase. Biochemical tests used for identification
of Streptococci Salt Tolerance Test Purpose: To
differentiate organisms positive for bile esculin are
separated into group D streptococci or Enterococcus
MEDIUM: 6.5% sodium chloride broth PURPOSE: •Used
to determine the effect of Optochin (ethyl
hydrocupreine hydrochloride) in an organism •Optochin
lyses Pneumococci (POSITIVE) Principle: •Optochin
interferes with the ATPase and production of ATP in
microorganisms Result: A zone of 14-16 mm is
considered Susceptible and presumptive identification
for Streptococcus pneumoniae. Optochin susceptibility
Biochemical tests used for identification of Streptococci
Bile Solubility •Confirmatory test for Streptococcus
pneumoniae •Used to differentiate S. pneumoniae from
Viridans strep Principle: Sodium desoxycholate (bile
salt) rapidly lyses pneumococcal colonies. Lysis depends
on the presence of an intracellular autolytic enzyme
Reagents: Plate (10% Sodium desoxycholate) Tube (2%
Sodium desoxycholate) Result: Tube: (+) No turbidity (-)
Remains Turbid Plate: (+) Lysed colonies (-) Intact
colonies