BACTERIOLOGY (LECTURE)

Streptococcus, Enterococcus, and Other LANCEFIELD'S CLASSIFICATION

Catalase-Negative, Gram-Positive Cocci - Developed by Rebecca Lancefield

GENERAL CHARACTERISTICS
● Streptococcus & Enterococcus spp. belong
to the family: Streptococcaceae
● Members of both genera are CATALASE (-),
GRAM (+) COCCI
● Smear: usually arranged in PAIRS or
CHAINS, cocci in pairs (lancet shaped)
● BAP: grayish, pinpoint with mucoid colonies ALPHA SCHEMATIC DIAGRAM
(due to their capsule)

CLASSIFICATION OF STREPTOCOCCI &

ENTEROCOCCI
● Smith & Brown Classification
- based on hemolytic reaction in BAP
● Lancefield’s Classification
- based on the presence of
serologically active carbohydrate
C-polysaccharide (cell wall CHO)
● Bergey’s Academic Classification
- Based on its thermal/ temperature
requirement

SMITH AND BROWN CLASSIFICATION

BETA SCHEMATIC DIAGRAM

● Alpha-hemolysis
- partial hemolysis of RBC
- greenish discoloration surrounding
bacterial colony
● Beta-hemolysis
- complete hemolysis of RBC
- clear zone of hemolysis surrounding
bacterial colony
NONHEMOLYTIC SCHEMATIC DIAGRAM
● Nonhemolytic
- old name: gamma hemolysis
- non-hemolytic
- no hemolysis of RBC
● Alpha prime
- wide zone hemolysis
- after colony, no hemolysis zone,
then wider zone of beta hemolysis

Tura, G. G.
 BACTERIOLOGY (LECTURE)

BERGEY'S ACADEMIC CLASSIFICATION c. BACTERIAL MENINGITIS

- seen in all age groups
- can follow when person has
pneumonia or otitis media

- SKIN TEST
- also known as FRANCIS TEST
Streptococcus pneumoniae - used to detect antibodies against
● a.k.a PNEUMOCOCCUS pneumococci
● Cell wall has C substance
● SBA: a hemolytic - VACCINES
● Young colonies: mucoid, DOME-SHAPED ● Pneumococcal Conjugate Vaccine 13
● Old colonies: DIMPLE SHAPED or DONUT (PCV13): for children
SHAPED (autolysis) - composed of purified polysaccharide
● Subculture 1-2 days to ensure viability conjugated to a Diphtheria protein
● Colonies may closely resemble colonies of - part of routine pediatric
VIRIDANS STREPTOCOCCI immunization
- 4 doses which begins 2 months of
- VIRULENCE FACTORS age
a. POLYSACCHARIDE CAPSULE ● Pneumococcal Polysaccharide Vaccine
- principal virulence factor for its 23 (PPSV23): for adults
pathogenesis - against 23 serotypes
- if capsule is removed: considered - recommended for adults older than
non-pathogenic 65 years old

- DISEASE ASSOCIATION - TREATMENT

a. BACTERIAL PNEUMONIA "LOBAR - Drug of choice: PENICILLIN
pneumonia"
- affects elderly and patients with
underlying diseases S. pneumoniae VS. Viridans
- for an individual to have the disease:
- organism is present in
nasopharynx
- deficient in antibody against
S. pneumonia
- not a primary infection; most of the
cases is due to the disturbance of 1. MOUSE VIRULENCE TEST
normal defence barrier in the body - strain is introduced on mouse
- nasopharynx > alveoli (S. - if mouse dies (S. pneumoniae), if mouse
pneumonia stimulate fluid; facilitator lives within 16-48 hrs (S. viridans
of the spread of organism) - only
stop when fluid reach fibrous septa;
separates the major lobes, hence
called lobar pneumonia
- most isolated: capsular serotypes 1,
2 and 3
- "rust-tinged" sputum
b. RECURRENT OTITIS MEDIA
- inflammation in middle ear
- usually affects children under 3 yrs.
old

Tura, G. G.
 BACTERIOLOGY (LECTURE)

- Equivocal:
- Any zone of inhibition <14mm is
2. INULIN FERMENTATION TEST questionable for pneumococci, the
- If bacteria ferment inulin strain will be confirmed using BILE
- Fermenter: S. pneumoniae SOLUBILITY
- Non-fermenter: S. viridans - QUALITY CONTROL
- Positive: Streptococcus pneumoniae
(ATCC6305)
3. BILE SOLUBILITY TEST - Negative: Streptococcus pyogenes
- Principle: (ATCC12384)
- Bile or a solution of bile salt (e.g.
sodium desoxycholate) rapidly lyses
pneumococcal colonies
- Reagent:
- 2% sodium deoxycholate
- If direct on agar: 10%; 1-2 drops
- Incubate: 10 minutes at 37C with
increased carbon dioxide; if
negative, reincubate for 2 hours
- Results
(+) Broth clearing 5. NEUFELD-QUELLUNG REACTION
(-) Turbid (CAPSULAR SWELLING)
- Results on Agar Plate - Principle:
- S. pneumoniae: colony disrupts - Test is performed by mixing on a
- S. viridans: remains as is slide a loopful of emulsified sample
with a loopful of
ANTI-PNEUMOCOCCAL SERUM
and METHYLENE BLUE
- Result:
(+) capsule appears SWOLLEN due
to a change in refractive index which
in turn is due to a serological
reaction (S. pneumoniae)
(-) No swell (S. viridans)
- Note: S. pneumoniae have capsule,
4. OPTOCHIN TEST (TAXO P) S. viridans if no capsule
- Principle:
- This test is used to determine the
effect of Optochin
(ethylhydrocupreine hydrochloride)
on an organism. Optochin lyses
pneumococci but not alpha
streptococci
- Optochin interferes with
ATPase and production of
ATP in the microorganism
- Expected Result:
(+) greater than or equal to 14mm
zone of inhibition- using 6mm disk
6. QUINIDINE
(SUSCEPTIBLE)
- (S) S. pneumoniae
(-) no zone of inhibition
- (R) S. viridans
(RESISTANT)

Tura, G. G.
 BACTERIOLOGY (LECTURE)

Viridans streptococci -Masks itself for it not to be

- Constituents of the normal microbiota of the recognized by the body
upper Respiratory Tract, female genital tract d. STREPTOLYSIN O (SLO)
and Gastrointestinal Tract - O2 labile, highly antigenic,
- Viridans: means green (refers to alpha hemolysis on SBA incubated
hemolysis exhibited by viridans) anaerobically
- Are fastidious organisms, with some strains - Lyse leukocytes, platelets, and other
requiring CO2 for growth cells (RBC)
- MOST COMMON ISOLATE: Streptococcus - Responsible for subsurface
mutans hemolysis (BAP)
- Groups: S. mitis group, S. mutans group, S. - Immunogenic
salivarius group, S. bovis group, S. - Test: Anti Streptolysin O Test (ASO);
anginosus group determine if individual has recent
- Most common cause of SUBACUTE infection with S. pyogenes (host will
BACTERIAL ENDOCARDITIS release antibody against antigen)
- Regarded as Opportunistic Pathogens (low e. STREPTOLYSIN S (SLS)
immunity or infections) - O2 stable, non-antigenic, hemolysis
- Others: Bloodstream infections, oral on SBA incubated aerobically
infections, meningitis, abscesses, - Lyse leukocytes, platelets, and other
osteomyelitis, and empyema (pus in pleural cells (RBC)
cavity) - Responsible for surface hemolysis
(BAP)
- Elaborated by serum
- Non-immunogenic
f. STREPTOKINASE
- Lysis of fibrin clots (plasminogen to
plasmin); plasmin lyse the fibrin clot
g. STREPTOCOCCAL PYROGENIC
Streptococcus pyogenes (Group A) EXOTOXINS (erythrogenic toxins)
- VIRULENCE FACTORS - Causes erythematous
a. M PROTEIN sandpaper-like rash "Scarlet Fever"
- Encoded by gene emm (200 - Superantigens: SpeA, SpeB, SpeC,
serotypes) and SpeF; stimulate T-cells resulting
- Emm1: most common serotype seen in the production of other
in pharyngitis components that will cause disease
- Attached to the peptidoglycan of the (immunogenic response); IL-I, TNF,
cell wall & extends to the cell Cytokines
surface
- Resist phagocytosis and play a role - DISEASE ASSOCIATION
in adherence of the bacterial cell to a. PHARYNGITIS or Tonsilitis: "STREP
mucosal cells THROAT"
- M1: most common serotype seen in - Most common clinical manifestation
pharyngitis of S. pyogenes
b. Lipoteichoic acid and protein F - Inflammation in pharynx and tonsils
(Fibronectin binding protein) - Most seen in children 5-15 years old
- Adhesion molecules - Incubation period: 1-4 days
- Used by bacteria to attach to cell - Symptoms: subside within 3-5 days
surface (unless if there is complications)
c. Hyaluronic acid capsule - Transmission: droplets and close
- Revents opsonized phagocytosis by contact
neutrophils or macrophages

Tura, G. G.
 BACTERIOLOGY (LECTURE)

- Diagnosis: throat culture, nucleic - Inflammation of

acid probe, amplification test, direct glomerulus and
antigen detection nephrons
- Essential step is proper - Immunologically
sampling mediated
- Create
b. POSTSTREPTOCOCCAL SEQUELAE antigen-antibody
- Complications if person have complex > immune
primary infection cause by S. complex > continue to
pyogenes circulate in the blood
- Sequelae: secondary > deposited in
1. Rheumatic Fever glomerulus >
- Follow after patient complement
has pharyngitis, inflammatory
inflammation of the response will damage
heart (joints, blood glomerulus
vessels, - Symptoms: puffy
subcutaneous tissue) face, and coca-cola
- Attack begin 1 month colored urine
after infection - Drug of choice: Penicillin
- Most serious result: Erythromycin (if
chronic progressive allergic to penicillin)
damage in heart
valve c. PYODERMAL INFECTIONS - skin
- Progress through 1. Impetigo
blood dissemination - small vesicles/pimples > turn
(antigenic cross to lesions; seen in 2-5 years
reactivity); antigen in old
S. pyogenes is the - inoculation of
same in heart tissue organism is
- Antibody is against through minor
emm protein and abrasions,
other antigen of S. insect bites
pyogenes, there are 2. Cellulitis
studies that that - inflammation of vessels
composition is the - deeper
same with heart invasion of
tissue, if body Strep
produce antibody - can be life
against that antigen threatening
(malibog siya) will if there's
create "antigenic bacteremia
cross reactivity" in the or sepsis
heart > antibody will 3. ERYSIPELAS
attack leading to - skin and
rheumatic fever subcutaneous
2. Acute Glomerulonephritis: tissue infection
"BRIGHT'S DISEASE" 4. Wound infection
- Most common in - dermal
children infection

Tura, G. G.
 BACTERIOLOGY (LECTURE)

5. SCARLET FEVER (SCARLATINA) and necrosis of skin (as well as

- Appears 1-2 after infected subcutaneous fat and fascia)
with bacteria - Life threatening
- Cardinal Signs: diffuse red - Acquired when individual experience
rash on the upper chest & trauma, burns, laceration
spreads to trunk & - Categorized by type 1, 2, and 3
extremities AND "strawberry - Type 1: polymicrobial
colored tongue" infection
- Type 2: only caused by S.
pyogenes
- Type 3: gas gangrene;
caused by Clostridium
- Other names:
● Galloping gangrene
- Rash will disappear 5-7 days ● Flesh eating bacteria
- Virulence: "Spe" cause syndrome
proliferation of T-Cell ● Suppurative fasciitis
- Susceptibility Test: ● Hospital gangrene
● DICK'S TEST - used ● Necrotizing erysipelas
to detect Erythrogenic
Toxin
(+) ERYTHEMA/
REDNESS ON SITE

e. STREPTOCOCCAL TOXIC SHOCK

SYNDROME
- A condition in which the entire organ
system collapses
- Portal of infection: unknown, others
from minor injury then complicate
- Produce a streptococcal pyrogenic
exotoxin - SpeA; play a major role in
- Diagnostic Test:
pathogenesis of TSS because they
● SCHULTZ- CHARLTON
are superantigen
TEST - used to diagnose
- Streptolysin O can contribute to toxic
whether the skin rashes are
shock
due to scarlet fever or not
- Inject anti-toxin into
- LABORATORY DIAGNOSIS
test arm
● On Sheep Blood Agar: a small,
- Neutralization of
transparent, and smooth with a well-
erythrogenic toxin
defined area of ẞ-hemolysis
(+) "BLANCHING
● Gram stain: gram-positive cocci
PHENOMENON" fading of
rashes

d. NECROTIZING FASCIITIS
- An invasive infection characterized
by fast progression of inflammation

Tura, G. G.
 BACTERIOLOGY (LECTURE)

● Lancefield Type Serologic Method can - QUALITY CONTROL

also be used if laboratory have ● Positive: Enterococcus
- Rapid identification test faecalis (ATCC29212)
● Other test: ● Negative: Streptococcus
○ Bacitracin Test agalactiae (ATCC10386)
○ PYR Test

- IDENTIFICATION TESTS
a. BACITRACIN TEST (TAXO A)
- A presumptive test which
differentiates Streptococcus
pyogenes from other Streptococci
- Principle: Based on the selective
inhibition of the growth of
S.pyogenes by a paper disc
containing 0.04 UNITS OF
BACITRACIN
- Mode of action: to inhibit the
synthesis of bacterial cell wall
- Result: Streptococcus agalactiae (Group B)
(+) zone of inhibition greater - VIRULENCE FACTORS
than 10 mm (S. pyogenes; a. Capsule made of SIALIC ACID
susceptable) - Most important virulence factor
(-) no zone of inhibition - Prevents phagocytosis
- Ineffective if opsonization
b. CAMP FACTOR (PROTEIN B)
- Pore forming protein secreted by the
organism

- DISEASE ASSOCIATION
- Most common etiologic agent of
NEONATAL SEPSIS and NEONATAL
MENINGITIS
- Most infections of infants occur in
the FIRST 3 DAYS AFTER BIRTH,
usually WITHIN 24 HOURS
- QUALITY CONTROL
- Most associated complication:
● Positive: Streptococcus
obstetric complication, rupture of
pyogenes (ATCC19615)
membranes, premature birth
● Negative: Streptococcus
A. Early-onset infection
agalactiae (ATCC27956)
- Babies less than 7 days old
- Manifest through pneumonia
b. PYR TEST (L-Pyrrolidonyl Arylamidase
and sepsis
Test)
- Mostly caused vertical
- Detect the organisms ability to
transmission (mother to
hydrolyze the substrate
child)
L-pyrrolidonyl-beta-napthylamide
- Most important factor:
- Results:
presence of bacteria in the
(+) Bright red color within 5
vagina of mother
min (S. pyogenes)
- It is recommended that all
(-) No color change
pregnant women will be

Tura, G. G.
 BACTERIOLOGY (LECTURE)

screened for S. agalactiae - LABORATORY DIAGNOSIS

35-37 weeks of gestation ● on Sheep Blood Agar : a grayish white
B. Late-onset infection mucoid colonies surrounded by a small
- Babies 7 days old to 3 zone of ẞ-hemolysis
months old ● Gram stain: gram-positive cocci; they also
- Manifest through meningitis form short chains (sample), longer chain
and sepsis (culture)
- Organism is rarely found in ● Presumptive identification: antigen
the vagina of mother before detection, lancefield, biochemical reaction
birth but since late ● Other test
- Detection: swab in vagina or ○ Hippurate Hydrolysis
rectum 35-37 weeks of ○ CAMP Test
gestation
- CDC recommends screening
during late 3rd trimester (If
positive, they are give
prophylactic antimicrobial
therapy; as well as mothers
with positive with urine
culture, and history with the
disease)
- 2 GBS patient types:
1. Healthy women that ● S. agalactiae should be inoculated in
generate infection SELECTIVE BROTH and CULTURE
after childbirth or MEDIA such as:
abortion (common a. TODD-HEWITT BROTH: 10 µg/mL
manifestation: wound COLISTIN and 15 µg/mL NALIDIXIC
infection, & ACID
endmatriitis) - Broth with 8 µg/mL
2. Elderly that is GENTAMICIN and 15 µg/mL
immunodeficient NALIDIXIC ACID (TransVag
(skin, soft tissue broth, Remel, Lenexa, KS)
infection, bacteremia can also be used
- Drug of choice b. StrepB Carrot Broth (SCB):
a. Penicillin produced ORANGE or RED
b. Combination of PIGMENT even after 6 HOURS OF
ampicillin and an INCUBATION
aminoglycoside (if
less susceptible to - IDENTIFICATION TESTS
penicillin) a. HIPPURATE HYDROLYSIS
- Use to determine whether a
microorganism, by action of the
enzyme HIPPURICASE
(HIPPURATE HYDROLASE) can
hydrolyze hippuric acid to benzoic
acid & glycine
- RESULTS:
(+) Deep purple color (S.
agalactiae)
(-) Colorless

Tura, G. G.
 BACTERIOLOGY (LECTURE)

Enterococcus spp. (Group D)

- Consists of gram (+) cocci that are natural
inhabitants of the intestinal tracts of humans
- QUALITY CONTROL: & animals
● Positive: Streptococcus - Subdivided into Enterococcal and
agalactiae (ATCC12386) Non-Enterococcal Groups (before)
● Negative: Streptococcus - Enterococcal Group: organism in
pyogenes (ATCC19615) intestinal tract; was placed in a new
genus called Enterococcus spp.
b. CAMP (Christie, Atkins & - Non-Enterococcal Group: remain in
Munch-Petersen) TEST group D Streptococci
- Differentiate S.agalactiae from other - Commonly identified species in clinical
Streptococci specimens: Enterococcus faecalis and
- PRINCIPLE: Enterococcus faecium
- CAMP factor is produced by - Unique because they have the ability to
S.agalactiae which has the GROW in EXTREME CONDITIONS (high
ability to act synergistically salt concentrations (6.5% NaCl), 45C, and
with the beta-hemolysin alkaline pH (some are resistant to
produced by S.aureus to antimicrobial agents)
produce even more potent - Frequent causes of NOSOCOMIAL
hemolysis. INFECTIONS - UTI and bacteremia
- PERFORMED IN THREE WAYS: ● UTI: most common; often associated
1. Use of β-lysin–producing with urinary catheterization or other
strain of S. aureus urologic manipulations
2. Use of a disk impregnated ● Enterococcal Bacteremia: prolonged
with the β-lysin hospitalization
3. Rapid CAMP Test (or Spot - Routinely used culture: Trypticase soy or
Camp Test): involves placing brain-heart infusion agar supplemented with
a drop of extracted β-lysin 5% sheep blood
- RESULT:
(+) "ARROW HEAD"
SHAPED ZONE OF
ENHANCED HEMOLYSIS
(-) No enhancement of
hemolysis
- QUALITY CONTROL:
- Positive: Streptococcus
agalactiae (ATCC13813)
- Negative: Streptococcus
pyogenes (ATCC19615)

Tura, G. G.
 BACTERIOLOGY (LECTURE)

b. SALT TOLERANCE TEST

- Used to determine the ability of an
organism to grow in high
concentrations of salt (6.5% NaCl)
- The salt tolerance test is a selective
and differential medium
- Enterococci are resistant to high salt
concentration
- A heart infusion broth containing
6.5% NaCl is used as the test
medium. This broth also contains a
small amount of glucose and
bromocresol purple as the indicator
- IDENTIFICATION TESTS for acid production
a. BILE ESCULIN TEST - RESULTS:
- Gram-positive bacteria other than (+) Visible turbidity in the
some streptococci and enterococci broth, with or without a color
are inhibited by the bile salts in this change from purple to yellow
medium (-) No turbidity and no color
- Organisms capable of growth in the change
presence of 4% bile and able to - QUALITY CONTROL:
hydrolyze esculin to esculetin will - Positive: Enterococcus
demonstrate growth faecalis (ATCC29212)
- Esculetin reacts with Ferric - Negative: Streptococcus
Ammonium Citrate (Fe3+) and gallolyticus (ATCC9809)
forms a dark brown to black
precipitate
- RESULTS:
(+) Growth and blackening of
the agar slant
(-) Growth and no blackening
of medium; No growth
- QUALITY CONTROL:
- Positive: Enterococcus
faecalis (ATCC19433)
- Negative: Streptococcus
pyogenes (ATCC19615)

Tura, G. G.
 BACTERIOLOGY (LECTURE)

SCHEMATIC DIAGRAM OF WHOLE GRAM

POSITIVE STREPTOCOCCI

References:
● Mahon, C. R., & Lehman, D. C. (2019).
Textbook of Diagnostic Microbiology (6th
ed.). Elsevier Inc.
● Tille, P. M. (2022). Bailey & Scott's
Diagnostic Microbiology (15th ed.). Elsevier,
Inc.
● Rodriguez, M. T. T. (2022). Review
Handbook in Diagnostic Microbiology (3rd
ed.). C & E Publishing Inc.

Tura, G. G.