Streptococcus, Enterococcus and Other Catalase-

Negative Gram-Positive Cocci

General Characteristics  Nonhemolytic
Streptococcus and Enterococcus spp. • No lysis of red blood cells around colony
 GPC in pairs and in chains (can be • No change in agar
encapsulated)
• Group A, B, and C (in young cultures),
Streptococcus pneumoniae  Alpha-prime (α ’)
 Ferments sugar except for manitol and sorbitol • Small area of intact red blood cells around
 Non motile except for group D colony
 Group A Streptococci have pili • Surrounded by a wider zone of complete
 They are more elongated than spherical hemolysis; Viridans group
 Catalase test (-) distinguishes from micrococci
and Staphylococci Classification of Streptococci and Enterococci
 Weak false-positive catalase reactions on media
containing blood - due to peroxidase activity of
hemoglobin
 Appear in chains when grown in both cultures
 Facultative anaerobes except for Viridans
streptococci that are microearophilic non-motile
 Some species are capnophilic
 Some species is stimulated by increase on CO2
but CO2 is not required
 Distribution
• Skin and mucous membranes
• Gastrointestinal
• Genitourinary Tract (depends of species)

Classification of Streptococci
Classification of Streptococci
Lancefield Classification
Academic or Bergey’s Classification
Lancefield’s (Groups A - G)
Clinically isolated Streptococci are divided into:
 C - substance or C carboyhydrates (cell wall
 Pyogenic streptococci
polysaccharides)
 Non-pyogenic streptococci
 Except for Viridans group and Streptococcus
Pyogenic streptococci isolated frequently from
pneumoniae
humans include:
 Agglutination test
 Streptococcus pyogenes
• Serologic group designated by letters
 Streptococcus agalactiae
 Developed by Rebecca Lancefield in 1930
 Streptococcus dysgalactiae subsp. Equisimilis
 Only 5 are significant to humans
 Streptococcus anginosus
• 3 of these pathogens have lancefield
Base on physiologic properties
antigens: Lancefield group A, B, and D.
1. Pyogenic Group
• The other two pathogenic species of the
• Grows at neither 10°C nor at 45°C
streptococcal genus do not have
2. Viridans Group
Lancefield antigens, therefore they are
• Grows at 45°C but not at 10°C
just called by their species name:
 Streptococcus mutans(causes
Streptococcus viridans
dental plaque)
Streptococcus pneumoniae
 Streptococcus saguis
 Streptococcus anginosus
Smith & Brown’s (hemolytic pattern)
 Streptococcus mitis
Types of Hemolysis
 Streptococcus salivarius
 Alpha (α )
3. Enterococcus group
• Partial lysis of red blood cells around colony
• Grows at both 10°C and 45°C but can
• Greenish discoloration of area around the
withstand temperature 60°C
colony
 Part of the normal flora
 Example:
Streptococcus(Enterococcus)
faecalis
 Beta (β )
4. Lactic Group
• Complete lysis of red blood cells around the
• Grows at 10°C but not at 45°C
colony
 Example: Streptococcus lactis
• Clear area around the colony

Fheb BMLS 2 - E
 Streptococcus, Enterococcus and Other Catalase-
Negative Gram-Positive Cocci
• responsible for sub-surface hemolysis in
β hemolytic Streptococci SBA
Group A Streptococcus(Streptococcus pyogenes) • lyses leukocytes, platelets, RBCs
 Antigenic Structure • antibodies measured by - anti-streptolysin O
• Lancefield group A (ASO)
• M protein  Hyaluronic acid capsule
 Attached to peptidogylcan of the cell • Responsible for the resistance to
wall phagocytosis (encoded by emm genes)
 Essential for virulence • Breakdown of connective tissues
 Colonizes
• Throat  adhesins
• Skin of humans  Streptokinase
Virulence Factors • lysis fibrin clots
 M Protein
• encoded by the genes emm
• major virulence factor, found in organism's
cell wall
• without M protein, it is avirulent
• Causes streptococcal cell to evade Clinical Infections
phagocytosis  Bacterial Pharyngitis
• play a role in adherence of the bacterial cell • Mot common infection caused by Group A
to mucosal cells Strep
• resistance to infection with Streptococcus • Spread by droplets and close contacts
pyogenes - related to the presence of • Diagnosis of streptococcal sore throat relies
type-specific antibodies to M protein on throat culture and direct antigen
• M1 serotype - most common serotype in detection
pharyngitis  Skin or Pyodermal Infection
 Dnases • Strains with temperature bacteriophage T12
• Help destroy foreign DNA by excreting it produce streptococcal pyogeneric
into surrounding cells exotoxins
 Lipoteichoic acid and Protein F • Diffuse red rash on the upper chest, trunk
• mediate adherence to host epithelial cells and extremitis
• Lipoteichoic acid - affixed to proteins on • Desquamation follows after rashes
the bacterial surface, together with M disappears
proteins  Scarlet Fever
• Protein F - secures the attachment of • Dick’s Test - susceptibility test for scarlet
streptococci to the oral mucosal cells fever
 Streptococcal pyrogenic exotoxin • Schultz-Charlton reaction (Blanching
• causes red spreading rash in scarlet fever phenomenon)
• Erythrogenic toxin  Neutralization of erythrogenic toxins
• Produced by lysogenic strains ( S. when an antitoxin is injected into
pyogenes infected with bacteriophage T12 skin of patient with scarlet fever skin
 Streptococcal pyrogenic exotoxin (SPE) rashes fades or blanch(+)
• Four immunologically distinct exotoxin types  Used to diagnosed when skin
found in Streptococcus pyogenes which rashes is due to scarlet fever or
acts as superantigens not
 SpeA SpeC  Necrotizing Fascitis(NF)
 SpeB SpeF • Invasive infection characterized by rapidly
• Causes a red spreading rash progressing inflammation and necrosis of
 Streptolysin S the skin, subcutaneous fat and fascia
• Non-antigenic • Life threatening infection
• oxygen stable (surface) • “flesh eating disease”
• responsible for surface hemolysis in SBA • Necrotizing fascitis may be categorized
(aerobic) as:
• not inhibited by antibody  NF type 1 - polymicrobial infection
 Streptolysin O from which aerobic and anaerobic
• Highly antigenic bacteria are recovered
• oxygen labile (active under anaerobic ◦ NF variant Type 1 - salt water NF
conditions) - Vibrio spp.

Fheb BMLS 2 - E
 Streptococcus, Enterococcus and Other Catalase-
Negative Gram-Positive Cocci
 NF Type 2 - only GAS  Anti-streptokinase
 NF Type 3 - gas gangrene or  Anti-hyaluronidase titers
clostridial myonecrosis
 Toxic Shock Syndrome Group B Streptococcus(Streptococcus agalactiae)
• Condition in which the entire organ systems Antigenic Structure
collapses, leading to death  Lancefield group B - specific antigen
• GAS associated with streptococcal TSS - • Acid-stable polysaccharide located in the
produce streptococcal pyrogenic exotoxin, cell wall
SpeA Colonizes
 Pyrogenic exotoxins functions as  Vaginal tract
superantigen  Can be normal respiratory flora in rare to few
 Oversimulation of immune response amounts
• SLO and various cell wall antigens - Virulence Factor
contribute to toxic shock  Capsule
 Post-streptococcal Sequelae • Prevents phagocytosis
• Rheumatic Fever • Ineffective after opsonization
 Follows streptococcus pyogenes • Sialic acid
pharyngitis  Most significant component of
 Most serious result in chronic, capsule
progresssive damage of the heart  Critical virulence determinant
valves  Hemolysin
 Acute RF and chronic sequela -  CAMP factor
rheumatic heart disease  Dnases
• Acute glomerulonephritis  Hyaluroidase
 After a cutaneous or pharyngeal  Protease
infection Clinical Infections
 Inflammatory reasponse causes  Invasive Disease in the Newborn
danage to glomeruli • Early-onset (less than 7 days old)
 Resulting to impairment of kidney  Most common,caused by vertical
function transmission from the mother
Susceptibility Testing  Colonization of the vagina and rectal
 GAS - susceptible to penicilin area
- sensitivity test are not routinely  Presents as a pneumonia or
performed meningitis with bacteria
 Erythromicin - penicilin allergic patients  Recommend all pregnant women be
Culture screened for____________
 SBA plate at 35°C streptococci at 35-37 weeks
 Ambient air or anaerobic conditions gestation
 Selective Media • Late-onset (atleast 7 days old)
• SBA containing sulfamethoxazole(SMZ) or  Usually presents as meningitis
colistin and polymixin B  Two types of patients in adults
• Improves recovery of β-hemolytic • Young previously healthy woman who
streptococci from throat cultures becomes ill after childbirth or abortion
Laboratory diagnosis  Endometritis
 Colonial morphology  Wound infectio
 Small, glossy, transparent, smooth, large  Endocarditis
zone beta hemolysis • Elderly person with serious underlying
Identification disease immunodefficiency
 PYR - positive (cannot be relied on solely for Susceptibility testing
identification) • Group B Streptococcus is still considered
 Bacitracin - sensitive; Presumptive test w/c susceptible to penicilin G
differetiates GAS from GBS • Sensitivity testing are not routinely
 SXT - resistant performed
 Sodium hippurate - Negative Laboratory diagnosis
 CAMP test - Negative  Colonial morphology
 Lancefield Typing - Group A Strep  Grayish, white, mucoid colonies
Immunologic tests to detect previous infection  Small zone of β - hemolysis
 Anti-Streptolysin (ASO)
 Anti-DNase B (most specific)

Fheb BMLS 2 - E
 Streptococcus, Enterococcus and Other Catalase-
Negative Gram-Positive Cocci
Culture • Opsonization of the capsule renders
 Detection of GBS in pregnant women organism avirulent
• Vaginal and rectal swab - 35-37 weeks of Clinical Infections
gestation  To cause disease: pneumococcal pneumonia
 35°C for 18-24 hours • Must be present in nasopharynx
 Subculture to SBA • Must be deficient in the specific circulating
 Selective Broth antibody against the capsular type strain
• Todd-Hewitt broth containing: of Streptococcus pneumoniae
 Colistin • Not a primary infection - disturbance of the
 Nalidixic acid normal defence barriers
Identification • Lobar pneumonia
 PYR - negative  Chills, cough, dyspnea (shortness of
 Sodium hippurate - positive breath)
 Bacitracin - Resistant  Leads to edema of the lungs and
 SXT - Resistant drowning in own fluids
 CAMP - positive  High mortality even with treatment
 Lancefield - group B strep (5% to 10%)
• Sinusitis
Schematic diagram for differentiation of GAS from • Otitis media
GBS  Both from children under 3 years old
• Bacteremia v
 Meningitis
 Both follow the previous infections,
creating spread over a wide area of
the body
• Can also be involved in
 Endocarditis
 Peritonitis
- All from dissemination
 Hemolytic uremic syndrome
- Blood and cerebrospinal fluid
(CSF) positive
- False positive Coombs test
often observed
Laboratory Diagnosis
 gram positive cocci in pairs (lancet shaped)
Group C and G Streptococci
 some requires increased CO2 - primary
Subdivided into large- colony and small-colony forms:
isolation
 Large-colony-forming β -hemolytic group C
 CULTURE MEDIA
and G
• BHI agar
• Streptococcus dysgalactiae subsp.
• TSA with 5% sheep RBCs
Equisimilis
• CHOC
 Small-colony-forming β -hemolytic group C
Cultural Characteristics
and G
 Enzyme - L-alanine-muramylamidase
• Streptococcus anginosus
• causes autolysis of cells
• produced upon prolonged incubation which
α hemolytic streptococci
causes the sunken center
Streptococcus pneumoniae
Antigenic Structure • checker or mucoid colonies
 C substance - antigen in cell wall  Large zone of a hemolysis on SBA
 Young cultures: round, glistening,wet
• C - reactive protein(CRP), β-globulin in
Presumptive identification
human serum reacts with C substance to
 Optochin (Taxo P)
form precipitate(chemical rxn)
• Sensitive
• CRP increases in inflammation and infection
Virulence Factor • Other a hemolytic species are resistant
 Capsular polysaccharide(capsular antigen) • Commonly used
• Capsule is antigenic  Bile solubility
• Positive
• Identified with Neufeld test (Quellung rxn)
• lysis of Streptococcus pneumoniae in the
presence of bile salt

Fheb BMLS 2 - E
 Streptococcus, Enterococcus and Other Catalase-
Negative Gram-Positive Cocci
Viridans Group Clinical infection
5 groups assignment of viridans group  UTIs
1. Streptococcus mitis group • Associated with urinary catheterization
2. Streptococcus mutans group • Other urologic manipulations
3. Streptoccocus salivarus group  Bacteremia
4. Streptoccocus bovis group
5. Streptoccocus anginus group Identification
 Catalase test
Group D Streptococci • Weak bubbling
Subdivided into:  PYR
 Enterococcal group • positive
• found in the intestinal tract  Bile esculin
• grows in nutient broth with 6.5% NaCl • positive
• bile esculin (+)  Growth in 6.5% Nacl
• now placed in genus Enteroccocus • positive
 Non-enterococcal group
• will not grow in nutrient broth with 6.5% Laboratory Diagnosis
NaCl  Routine culture media
• bile esculin (+) • TSA or BH| agar with 5% sheep blood
Virulence Factor • grows at 35°C in CO,
 Anginus group  Selective media containing:
• polysaccharide capsule and cytolysin  bile esculin azide
• Extracellular dextran  colistin-nalidixic acid
• cell surface-Associated proteins (adhesin)  phenylethyl alcohol
 Group C and G  chromogenic substrates
• M protein • Cephalexin-aztreonam-arabinose agar
• extracellular enzymes  Enterococcus spp. are Mentified based on:
Laboratory Diagnosis a. ability to produce acid in carbohydrate broth
 PYR b. ability to hydrolyze arginine
• All members are negative c. Tolerance of 0.04% tellurite
• Separates group D from Enetrococcus d. utilization of pyruvate
 LAP (leucine amino peptidase) e. ability to produce acid from methyl-a-D-
• All members are positive glucopyranoside
 Optochin (Taxo P) f. growth around 100-ug efrotomycin acid disk &
• Resistant motility
 Bile solubility Classification schemes of Streptococcace
• Negative 1. Hemolytic pattern on SBA
 Bile esculin 2. Physiologic characteristics
• Negative a) Pyogenic
 Growth in 6.5% NaCl  produce pus
• No Growth  B-hemolytic
 most Lancefield groups
Enterococcus b) Lactococci
 Includes:  nonhemolytic
• Enterococcus faecalis  Lancefield group N antigen
• Enterococcus faecium  often found in dairy products
 All species produce cellwall-associated group D c) Enterococci
antigen  normal microbiota of human
 Normal flora of: intestines
• intestine and mucous membranes but d) Viridans
causes endogenous and exogenous  Normal microbiota of the upper
disease states respiratory tract of humans
 Grows in extreme condition  Lack a C carbohydrates
 Resistant to multiple antibiotics  Not part of the Lancefield
classification
Susceptibility testing  Opportunistic pathogens
 Vancomycin resistant strains of Enterococcus 3. Serologic groupings or typing of C carbohydrates,
species exist should be performed only if the capsular polysaccharide, or surface protein (M protein)
isolate is clinically significant

Fheb BMLS 2 - E
 Streptococcus, Enterococcus and Other Catalase-
Negative Gram-Positive Cocci
Biochemital characteristics • ELISA (Enzyme linked
Bacitracin susceptibility test immunosorbent assay)
 presumptive identification of Streptococcus Nucleic acid Probe
pyogenes
 Screening of Streptococcus pyogenes of GAS in
throat cultures
 SBA containing SMZ and a bacitracin disk
placed on the agar
• only S. pyogenes and S. agalactiae grow

PYR (pyrrolindonyl-α-Naphthylamide hydrolysis)

 presumptive identific qation of the B-
hemolytic GAS and the nonhemolytic group D
 more specific for Streptococcus pyogenes than
Bacitracin
 Streptococcus pyogenes
• only species of Streptococcus that is PYR
(+)

CAMP test (Christie, Aikins & Munch-Peterson)

 presumptive identification of G3*;
 performed in 3 ways:
• use of B-lysin-producing strain of
Staphylococcus aure
• use of disk impregnated with the B-lysin
• rapid CAMP test

Hippurate hydrolysis
 Differentiates Streptococcus agalactiae from
other B-hemolytic streptococci
 hippuricase hydrolyzes sodium hippurate to
form sodium benzoate and glycine

Voges-Proskauer test
 distinguish small-colony-forming B-hemolytic
anginosus group containing groups A and C
antigens from large-colony-forming pyogenic
strains with the antigens

Optochin Susceptibility
 Optochin - ethylhydrocuprein hydrochloride
 Presumptive identification of Streptococcus
pneumoniae
 Isolates producing smaller colonies - confirms
identity by bile solubility test

Noncultural Identification
Immunoassay
 GAS - detection of the group-specific antigen
- colonies or throat swab
• Slide agglutination

Fheb BMLS 2 - E