MLAB 2434: Microbiology
Keri Brophy-Martinez
Streptococci, Enterococci and Other
Catalase-Negative Gram Positive Cocci
Streptococcus and Enterococcus:
General Characteristics

Members of the Streptococcaceae family

Facultatively anaerobic

Aerotolerant

Catalase negative
Streptococcus and Enterococcus:
General Characteristics


Most are typically spherical; some may appear elongated or ovoid
They may appear in chains or pairs
Streptococcus and Enterococcus:
Habitat and Clinical Infections

Habitat
◦ Normal Flora
 Respiratory tract
 Gastrointestinal tract
 Urogenital tracts

Clinical Infections
◦ Upper and lower
respiratory tract
infections
◦ Urinary tract
infections
◦ Wound infections
◦ Endocarditis
Streptococcus and Enterococcus:
Cell Wall Structure

Thick peptidoglycan layer

Teichoic acid

Carbohydrate layer present
◦ Used in Lancefield grouping of
Streptococcus spp.

Capsule
◦ Virulence factor
◦ S. pneumoniae
Classification Overview

Physiologic characteristics
◦ Pyogenic: produce pus
◦ Lactococci: found in dairy products
◦ Enterococci: normal gut flora
◦ Viridans: normal URT flora

Hemolysis
◦ J. H Brown
◦ Alpha, beta, gamma classifications

Serological grouping
◦ Typing of C carbohydrate
◦ Lancefield group
◦ Performed only on β-hemolytic hemolysis

Biochemical
◦ Based on reaction of isolate
Classification:
Hemolysis
J.H. Brown- 1903
 Grouped streps on ability to lyse RBCS

◦
◦
◦
◦
Alpha
Beta
Gamma
Alpha-prime
Hemolysis Patterns
◦ Alpha (α):
 Greenish
discoloration
 Caused by partial
lysis of RBCs in
media
Hemolysis Patterns

Beta (ß):
◦ Complete lysis of RBCs
◦ Produces a clear, colorless
zone
Hemolysis Patterns
◦ Gamma :
 Colonies show
no hemolysis or
discoloration
 Called nonhemolytic
Classification:
Serological Grouping
Rebecca Lancefield – 1930
 Based on presence of carbohydrates in
cell wall
 Groups A, B, C, and D most significant
 Typing done on beta-hemolytic
colonies

Classification:
Biochemical Identification/Susceptibility

Bacitracin
◦ “A” disk or “Taxo A” disk
◦ 0.04 units
◦ Identifies Group A
streptococci (S. pyogenes)
◦ Zone of inhibition is
presumptive ID of Grp. A
strep
Group A streptococcus is susceptible
to “A” disk (left)
Biochemical Identification/Susceptibility

Optochin
◦ P disk or“Taxo P” disk
◦ Differentiates S. pneumoniae from other
alpha-hemolytic streptococci
Biochemical Identification

Bile solubility test
◦ Detects amidase enzyme
◦ Under bile salt or detergent lyses cell wall
 Clearing through lysis of colonies
◦ Diagnostic for S. pneumoniae
Biochemical Identification

PYR hydrolysis
◦ Substrate L-pyrrolidonyl-bnapthlyamide (PYR) is
hydrolyzed by the enzyme
pyrrolidonyl arlamide
◦ Group A Streptococci and
Enterococcus sp. posses the
necessary enzyme.
◦ More specific than Bacitracin
for Group A streptococci
The disk on the right has turned
a red color, indicating a
indicating a positive reaction.
The left disk remains a yellow
color indicating a negative result.
Biochemical Identification

Hippurate hydrolysis
◦ Differentiates Group B streptococci from
other beta hemolytic streptococci
◦ Group B streptococci hydrolyzes sodium
hippurate forming a purple color
Biochemical Identification

CAMP test
◦ Christie,Atkins, MunchPetersen
◦ Detects the production of
enhanced hemolysis that
occurs when b-lysin and the
hemolysins of Group B
streptococci come in contact
with each other
Group B streptococci showing the
classical “arrow-shaped
hemolysis near the
staphylococcus streak
Biochemical Identification

Bile esculin
hydrolysis
◦ Ability to grow in bile
and hydrolyze Esculin
◦ Characteristic of
streptococci that
possess group D
antigen and
Enterococci
Both Group D streptococci
and enterococci produce a
positive (top) bile Esculin
hydrolysis test.
Biochemical Identification

Salt Tolerance
◦ Growth in 6.5% NaCl
broth
◦ Differentiates Group D
streptococci from
enterococci
◦ Enterococcus= POSITIVE
 Tube on left
◦ Group D Streptococcus=
NEGATIVE
 Tube on right
Non-culture Identification

Slide agglutination kits
◦ Latex beads are coated with group specific
anti-serum, which clump when mixed with a
small amount of colony from the specific
Streptococcus sp.

Nucleic Acid Probes
◦ Detect genes for specific groups
Slide Agglutination Tests
Slide Agglutination Tests
Break Time!
Virulence Factors:
Streptococcus pyogenes

Fimbrae: Protein F
◦ Attachment and adherence

M protein:
◦ Resistance to phagocytosis

Hyaluronic acid capsule:
◦ Prevents phagocytosis

Lipoteichoic acid:
◦ Adheres to molecules on host
epithelial cells
Virulence Factors:
Streptococcus pyogenes

Hemolysins
◦ Streptolysin O (O2 labile) detected in ASO titers
◦ Streptolysin S (O2 stable) Causes hemolysis on plates

Erythrogenic toxin/Streptococcal pyogenic
exotoxin:
◦ Scarlet fever

Enzymes
◦ Streptokinase
◦ DNases
◦ Hyaluronidase – “spreading factor”
Clinical Conditions:
Streptococcus pyogenes(Group A)
Clinical Conditions:
Streptococcus pyogenes(Group A)

Pyodermal infections
◦ Impetigo: weeping
lesion
◦ Erysipelas

Cellulitis

Wound Infections
Erysipelas due to
Streptococcus pyogenes
Clinical Conditions:
Streptococcus pyogenes(Group A)

Scarlet Fever
◦ Starts with pharyngitis and causes rash on trunk
and extremities
◦ Due to untreated Group A infections
Invasive Group A
Streptococcal Infections

Streptococcal toxic shock syndrome
◦ Multi-organ system failure similar to staphylococcal toxic
shock
◦ Initial infection may have been pharyngitis, cellulitis,
peritonitis, or other wound infections
Invasive Group A Streptococcal
Infections

Cellulitis/Necrotizing Fasciitis
◦ Severe form of infection that is life-threatening
◦ Bacteremia and sepsis may occur
◦ In patients necrotizing fasciitis, edema, erythema, and pain
in the affected area may develop
◦ Streptococcal myositis resembles clostridial gangrene
Post–Group-A
Streptococcal Infections

Rheumatic fever
◦ Fever
◦ Inflammation of the heart, joints, blood vessels, and
subcutaneous tissues
◦ Chronic, progressive damage to the heart valves
(most evidence favors cross-reactivity between Strep.
antigens and heart tissue)
◦ ASO titer will be elevated
Post–Group-A
Streptococcal Infections

Acute glomerulonephritis (AGN)
◦
◦
◦
◦
Follows either cutaneous or pharyngeal infections
More common in children than adults
Antigen-antibody complexes deposit in the glomerulus
Inflammatory response causes damage to the
glomerulus and impairs the kidneys
Laboratory Diagnosis:
Group A Streptococcus
Grams stained wound smear showing gram-positive
cocci in chains with numerous “polys” (PMNs)
Laboratory Diagnosis:
Group A Streptococcus

Colony morphology
◦ Transparent, smooth, and
well-defined zone of
complete or b- hemolysis
Laboratory Diagnosis:
Group A Streptococcus

Identification
◦
◦
◦
◦
Catalase-negative
Bacitracin-susceptible
PYR-positive
Hippurate hydrolysisnegative
◦ Slide agglutination
Group A streptococci is susceptible to
Bacitracin disk (left); The right shows
resistance
Group B b-Hemolytic Streptococcus
(Streptococcus agalactiae)
Colonize the urogenital tract of pregnant women
(10-30% rate – can cause OB complications such
as premature rupture of membranes and
premature delivery)
 Mother fails to pass protective antibodies to fetus
 Cause invasive diseases in newborns

◦ Early-onset infection
◦ Late-onset disease
Invasive Disease in the Newborn
Early Onset
Late-Onset
Age of Onset
< 7 days
7 – 30 days
Median age of onset
1 hour
27 days
Maternal complications of
labor
Common
Less common
Incidence of prematurity
25%
Less common
Source of Organism
Maternal genital tract
Maternal genital tract;
nosocomial; community
Clinical presentation
Nonspecific (35-55 %)
Meningitis 5-10 %
Respiratory diseases 3555 %
Focal
Meningitis 25-35 %
Types
I, II III, V
III (75%)
Mortality Rate
5-15 %
2-10 %
Invasive Streptococcus agalactiae
Infections

In adults
◦ Occurs in immunosuppressed patients or those with
underlying diseases
◦ Often found in a previously healthy adult who just
experienced childbirth
Laboratory Diagnosis:
Streptococcus agalactiae

Colony morphology
◦
◦
◦
◦
Small
Grayish-white
Mucoid, creamy
Narrow zone of b-hemolysis
Laboratory Diagnosis:
Streptococcus agalactiae

Presumptive Identification
tests
◦
◦
◦
◦
◦
Gram stain- GPC in chains
Catalase-negative
Bacitracin-resistant
Bile esculin- negative
Does not grow well in 6.5%
NaCl.
◦ CAMP- positive
◦ Slide agglutination
S. agalactiae shows the arrowshaped hemolysis near the
staphylococcus streak, showing a
positive test for CAMP factor
Streptococcus pneumoniae

General characteristics
◦ Inhabits the nasopharyngeal areas of healthy individuals
◦ Typical opportunist
◦ Possess C substance

Virulence factors
◦ Polysaccharide capsule
Clinical Conditions:
Streptococcus pneumoniae

Pneumonia
◦ Most common cause of bacterial pneumonia

Meningitis

Bacteremia

Sinusitis/otitis media
◦ Most common cause of otitis media in children < 3 years
Laboratory Diagnosis:
Streptococcus pneumoniae

Microscopic morphology
◦ Gram-positive cocci in
pairs; lancet-shaped
(somewhat oval in
shape)
Laboratory Diagnosis:
Streptococcus pneumoniae

Colony morphology
◦ Smooth, glistening,
wet-looking, mucoid
◦ a-Hemolytic
◦ CO2enhances
growth
◦ As colony ages,
autolytic collapse
causes “checker
shape”
Laboratory Diagnosis:
Streptococcus pneumoniae

Identification
◦ Catalase negative
◦ Optochin-susceptibilitytest–susceptible
◦ Bile-solubility-test–
positive
Identification Schema
Enterococcus Species

Clinically Significant Isolates
◦ E. faecalis
◦ E. faecium

Opportunistic pathogens
◦ In the GI tract, genitourinary tract and oral cavity

Associated infections
◦
◦
◦
◦
◦
Bacteremia
Urinary tract infections
Wound infections
Endocarditis
Hospital-acquired Infections
Laboratory Diagnosis:
Enterococcus Species

Microscopic morphology
◦ Cells tend to elongate

Colony morphology
◦ Small, grey
◦ Most are non-hemolytic,
although some may show
a- or, rarely, b-hemolysis
◦ Possess Group D antigen
Laboratory Diagnosis:
Enterococcus Species

Identification tests
◦ Catalase: may produce a weak catalase reaction
◦ Hydrolyze bile esculin
◦ Differentiate Group D from Enterococcus sp. with 6.5%
NaCl or PYR test
◦ Important to identify Enterococcus from non-Enterococcus,
because Enterococcus must be treated more aggressively.
Identification Schema
Or PYR disk
Other Streptococcal Species

Viridans group (Viridans means “green”)
◦ Members of the normal oral, nasopharyngeal flora, GI tract and female
genital tract
◦ Most are a hemolytic but also includes nonhemolytic species
◦ The most common cause of subacute bacterial endocarditis
◦ Also involved with gingivitis and dental carries
◦ PYR= negative
◦ Optochin= negative
◦ Bile solubility= negative
(SBE)
Viridans

5 groups
◦ Anginosus
 S. anginosus, S. intermedius, S. constellatus
◦ Mitis
 S. sanguig, S. parasanguis, S. gordonii, S. crista, S. infantis, S. mitis,
S. oralis, S. oralis, S. peroris
◦ Mutans
 S. criceti, S. downei, S. macacae, S. mutans, S. rattus, S. sobrinus
◦ Salivarius
 S. salivarius, S. thermophilus, S. vestibularis
◦ Bovis
 S. equinus, S. gallolyticus,S. infantarius, S. alactolyticus
Abiotrophia & Granulicatella
◦ Once referred to as Nutritionally variant
streptococci (NVS)
◦ Causes endocarditis and otitis media
◦ Normal flora of oral cavity
◦ Requires pyridoxal to grow (can satellite
around Staph, E. coli, Klebsiella, Enterobacter
and yeasts)
Streptococcus and Enterococcus
Species
S.pyogenes
ß
Group
Antigen
A
S.agalactiae
ß
B
Group B
streptococci
S. equisimilis
ß
C
E. faecalis
E. faecium
E. durans
Alpha or no
hemolysis
( rarely ß )
D
Group C
streptococci
Enterococci
S. bovis
S. equinus
Alpha (a)or
none
(rarely ß)
D
Nonenterococci
S. pneumoniae
Viridans and
Nonhemolytic
S. sanguis
S. salivarius
S. mitis or
nonhemolytic
S. milleri
S. mutans
Other species
Hemolysis
Alpha (a)
hemolysis
Alpha (a)
hemolysis or
no
hemolysis
-
Common
Terms
Group A
streptococci
Disease Association(s)
Pharyngitis; scarlet fever
pyoderma; rheumatic
fever; AGN
Neonatal sepsis;
puerperal fever;
pyogenic infections;
pneumonia; meningitis
Pharyngitis; impetigo;
pyogenic infections
Urinary tract infections
Wound infections
Bacteremia;
Endocarditis
Urinary tract; pyogenic
infections;
Endocarditis infections
Pneumococcus
Bacteremia;
pneumonia;
meningitis;
Viridans strep
Endocarditis
Dental caries
Streptococcus and Enterococcus

Treatment
◦ Generally, streps are not routinely tested for
susceptibility since penicillin drug of choice. If
the patient is allergic to pen use erythromycin.
◦ Antibiotic resistance seen with Enterococcus,
use vancomycin
References
http://archive.microbelibrary.org/ASMOnly/Deta
ils.asp?ID=2566
 http://www.goodtoknow.co.uk/health/Scarletfever
 http://onwardstate.com/2009/12/10/keep-yourgoals-to-yourself/
 Mahon, C. R., Lehman, D. C., & Manuselis, G.
(2011). Textbook of Diagnostic Microbiology (4th
ed.). Maryland Heights, MO: Saunders.
