Streptococci

Characters of Streptococci
– Gram positive cocci
– 1µm in diameter
– Chains or pairs
– Usually capsulated
– Non motile

– Non spore forming

– Facultative anaerobes
– Fastidious
– Catalase negative (Staphylococci are catalase positive)
The term streptococcus was first applied by
Billroth in 1874, to a chain forming
.coccus he saw in infected wounds
Pasteur and Rosenbach described its
morphology, cultural characters, and virulence
.for mice and rabbits
 Classification of Streptococci

Streptococci can be classified according to:

– Oxygen requirements
Anaerobic (Peptostreptococcus)
Aerobic or facultative anaerobic (Streptococcus)
– Serology (Lanciefield Classification)
– Hemolysis on Blood Agar (BA)
 Serology: Lanciefield Classification
Streptococci

Lanciefield classification

Group A Group B Group C Group D Other groups

S. pyogenes S. agalactiae S. equisimitis Enterococcus (E-U)

Streptococci classified into many groups from A-K & H-V

One or more species per group
Classification based on C- carbohydrate antigen of cell wall
– Groupable streptococci
A, B and D (more frequent)
C, G and F (Less frequent)
– Non-groupable streptococci
S. pneumoniae (pneumonia)
viridans streptococci
– e.g. S. mutans
– Causing dental carries
Classification of Streptococci Based on
Hemolysis on Blood Agar
Hemolysis on BA
 -hemolysis
Partial hemolysis
Green discoloration around the colonies

 -hemolysis
Complete hemolysis
Clear zone of hemolysis around the colonies
 -hemolysis
No lysis
Streptococci

-hemolysis -hemolysis -hemolysis

Hemolysis on Blood agar

-hemolysis

-hemolysis

-hemolysis
 Alpha-hemolytic

Pneumococci
– S. pneumoniae (sometimes called Pneumococcus),
is a leading cause of bacterial pneumonia and
occasional etiology of otitis media, sinusitis,
meningitis and peritonitis.
The Viridans group: alpha-hemolytic
– S. mutans, a contributor to dental caries
– S. mitis, mostly found around cheek region
– S. sanguinis, no preference of locations
– S. salivarius, mostly found on the dorsal side of the
tongue
– S. salivarius ssp. thermophilus, used in the manufacture
of some cheeses and yogurts
– S. constellatus, occasional human pathogen, notable as
colonies grown on blood agar smell strongly of caramel
 Beta-hemolytic

Group A
– S. pyogenes, also known as Group A
Streptococcus (GAS), is the causative agent in
Group A streptococcal infections, including
streptococcal pharyngitis ("strep throat"), acute
rheumatic fever, scarlet fever, acute
glomerulonephritis and necrotizing fasciitis. Strep.
pyogenes is the other major cause of streptococcal
infection in humans, after pneumococcus
Group B
– S. agalactiae, or GBS, causes pneumonia and
meningitis in neonates and the elderly, with
occasional systemic bacteremia. They can also
colonize the intestines and the female reproductive
tract, increasing the risk for premature rupture of
membranes and transmission to the infant.
Group C
– This group includes S. equi, which causes strangles
in horses,[10] and S. zooepidemicus
Group D (enterococci)
– Many former Group D streptococci have been
reclassified and placed in the genus Enterococcus
(including Enterococcus faecalis, Enterococcus
faecium, Enterococcus durans, and Enterococcus
avium).[11] For example, Streptococcus faecalis is
now Enterococcus faecalis.
Group F streptococci
– Group F streptococci were first described in 1934 by Long
and Bliss amongst the "minute haemolytic streptococci".[12]
They are also known as Streptococcus anginosus (according
to the Lancefield classification system) or as members of the
S. milleri group (according to the European system).
Group G streptococci
– These streptococci are usually, but not exclusively, beta-
hemolytic. Streptococcus canis is an example of a GGS
which is typically found on animals, but can cause infection
in humans.
 Group A streptococci

Include only S. pyogenes

Group A streptococcal infections affect all ages peak
incidence at 5-15 years of age
90% of cases of pharyngitis
Streptococcus pyogenes is a spherical, Gram-positive
bacterium that is the cause of group A streptococcal
.infections
S. pyogenes displays streptococcal group A antigen on
.its cell wall
S. pyogenes typically produces large zones of when
cultured on blood agar plates, and are therefore also
called Group A (beta-hemolytic) Streptococcus
.(abbreviated GABHS)
Streptococci are catalase-negative. In ideal
conditions, S. pyogenes has an incubation
period of approximately 1–3 days. It is an
infrequent, but usually pathogenic, part of the
skin flora.
It is estimated that there are more than 700
million infections each year and over 650,000
cases of severe, invasive infections that have a
mortality rate of 25 %.
Pathogenesis and Virulence Factors
Structural components
– M protein M, which interferes with opsonization and lysis of the
bacteria
– Lipoteichoic acid & F protein adhesion
– Hyaluronic acid capsule, which acts to camouflage the bacteria
Enzymes
– Streptokinases
– Deoxynucleases facilitate the spread of streptococci through tissues
– C5a peptidase
Pyrogenic toxins that stimulate macrophages and helper T
cells to release cytokines
Streptolysins
– Streptolysin O lyse red blood cells, white blood cells, and platelets
– Streptolysin S
 Disease caused by S. pyogenes
Suppurative
– Non-Invasive
Pharyngitis (“strep throat”)-inflammation of the pharynx
Skin infection, Impetigo
– Invasive
Scarlet fever-rash that begins on the chest and spreads across the
body
Pyoderma-confined, pus-producing lesion that usually occurs on the
face, arms, or legs
Necrotizing fasciitis-toxin production destroys tissues and eventually
muscle and fat tissue
Non Suppurative
– Rheumatic fever: Life threatening inflammatory disease that
leads to damage of heart valves muscle
– Glomerulonephritits
Immune complex disease of kidney
inflammation of the glomeruli and nephrons which obstruct blood
flow through the kidneys
 Post streptococcal sequelae
Infection with Streptococcus pyogenes can give rise •
to serious nonsuppurative sequelae: acute
.rheumatic fever and acute glomerulonephritis
These pathological events begin 1-3 weeks after an •
acute streptococcal illness, a latent period consistent
.with an immune-mediated etiology
Whether all S. pyogenes strains are rheumatogenic is •
controversial; however, clearly not all strains are
.nephritogenic
Acute rheumatic fever is a sequel only of
pharyngeal infections, but acute
glomerulonephritis can follow infections of
.the pharynx or the skin
Although there is no adequate explanation for
the precise pathogenesis of acute rheumatic
fever, an abnormal or enhanced immune
.response seems essential
Acute rheumatic fever can result in permanent
damage to the heart valves. Less than 1% of
sporadic streptococcal pharyngitis infections
result in acute rheumatic fever
Acute glomerulonephritis results from
deposition of antigen-antibody-complement
complexes on the basement membrane of
.kidney glomeruli
The antigen may be streptococcal in origin or
it may be a host tissue species with antigenic
determinants similar to those of streptococcal
antigen
Severe, sometimes life-threatening, GAS disease may occur
when bacteria get into parts of the body where bacteria
usually are not found, such as the blood, muscle, or the
lungs. These infections are termed "invasive GAS
“.disease

Two of the most severe, but least common, forms of

invasive GAS disease are necrotizing fasciitis and
.Streptococcal Toxic Shock Syndrome
Necrotizing fasciitis (occasionally described by the media -
as "the flesh-eating bacteria") destroys muscles, fat, and
skin tissue. Streptococcal toxic shock syndrome (STSS),
causes blood pressure to drop rapidly and organs (e.g.,
.kidney, liver, lungs) to fail

About 20% of patients with necrotizing fasciitis and more -

.than half with STSS die
About 10%-15% of patients with other forms of invasive -
.group A streptococcal disease die
Early signs and symptoms of
;necrotizing fasciitis

Fever -
Severe pain and -
Swelling -
Redness at the wound site -
Early signs and symptoms of
;STSS
Fever -

Dizziness -

Confusion -

A flat red rash over large -

areas of the body
 Differentiation between -hemolytic
streptococci

The following tests can be used to differentiate between

-hemolytic streptococci
– Lanciefield Classification
– Bacitracin susceptibility Test
Specific for S. pyogenes (Group A)
– CAMP test
Specific for S. agalactiae (Group B)
 Bacitracin sensitivity
Principle:
– Bacitracin test is used for presumptive
identification of group A
– To distinguish between S. pyogenes
(susceptible to B) & non group A such as S.
agalactiae (Resistant to B)
– Bacitracin will inhibit the growth of gp A
Strep. pyogenes giving zone of inhibition
around the disk
Procedure:
– Inoculate BAP with heavy suspension of tested
organism
– Bacitracin disk (0.04 U) is applied to
inoculated BAP
– After incubation, any zone of inhibition around
the disk is considered as susceptible
Susceptibility test
Trimethoprim sulfamethoxazole –
(SXT)
Inhibits beta-hemolytic
streptococcal groups other than
A and B

Group A streptococcus growing in the

presence of SXT
 Biochemical Identification
PYR hydrolysis
Substrate L-pyrrolidonyl- –
napthlyamide (PYR) is
hydrolyzed by Group A
Streptococci and
.Enterococcus sp
As specific as 6.5% NaCl –
.broth for Enterococcus sp
More specific than Bacitracin – PYR test for Group A streptococci
for Group A streptococci and enterococci. Both are positive
for this test (right); left is a negative
result
 Biochemical Identification

Hydrolysis
Hippurate hydrolysis –
Differentiates Group B streptococci from other beta hemolytic
streptococci
Group B streptococci hydrolyzes sodium hippurate
 Biochemical Identification
Christie-Atkins, Munch-
Petersen (CAMP) test
Detects the production of –
enhanced hemolysis that
occurs when -lysin and the
hemolysins of Group B
streptococci come in contact

Group B streptococci showing the

classical “arrow-shaped hemolysis
near the staphylococcus streak
 CAMP test
Principle:
– Group B streptococci produce extracellular protein (CAMP factor)
– CAMP act synergistically with staph. -lysin to cause lysis of RBCs

Procedure:
– Single streak of Streptococcus to be tested and a Staph. aureus are made
perpendicular to each other
– 3-5 mm distance was left between two streaks
– After incubation, a positive result appear as an arrowhead shaped zone of
complete hemolysis
– S. agalactiae is CAMP test positive while non gp B streptococci are
negative
CAMP test
Differentiation between -hemolytic
streptococci

The following definitive tests used to differentiate

between S. pneumoniae & viridans streptococci

– Optochin Test
– Bile Solubility Test
– Inulin Fermentation
Optochin Susceptibility Test
Principle:
– Optochin (OP) test is presumptive test that is used to identify S.
pneumoniae
– S. pneumoniae is inhibited by Optochin reagent (<5 µg/ml) giving a
inhibition zone ≥14 mm in diameter.
Procedure:
– BAP inoculated with organism to be tested
– OP disk is placed on the center of inoculated BAP
– After incubation at 37oC for 18 hrs, accurately measure the
diameter of the inhibition zone by the ruler
– ≥14 mm zone of inhibition around the disk is considered as positive
and ≤13 mm is considered negative
S. pneumoniae is positive (S) while S. viridans is negative
(R)
Optochin Susceptibility Test

Optochin resistant
S. viridans

Optochin susceptible
S. pneumoniae
 Bile Solubility test

Principle:
– S. pneumoniae produce a self-lysing enzyme to inhibit the
growth
– The presence of bile salt accelerate this process
Procedure:
– Add ten parts (10 ml) of the broth culture of the organism to be tested
to one part (1 ml) of 2% Na deoxycholate (bile) into the test tube
– Negative control is made by adding saline instead of bile to the culture
– Incubate at 37oC for 15 min
– Record the result after 15 min
 Bile Solubility test

Results:
– Positive test appears as clearing in the
presence of bile while negative test
appears as turbid
– S. pneumoniae soluble in bile whereas
S. viridans insoluble
Laboratory Diagnosis:
Group A Streptococcus

Grams stained wound smear showing gram-positive cocci

”in chains with numerous “polys
 Laboratory Diagnosis:
Group A Streptococcus
Colony morphology
Transparent, smooth, and –
well-defined zone of complete
or- hemolysis
 Laboratory Diagnosis:
Group A Streptococcus
Identification
Catalase-negative –
Bacitracin-susceptible –
PYR-positive –
Bile-esculin–negative –
NaCl-negative 6.5% –

Group A streptococci is susceptible to

Bacitracin disk (left); The right shows
resistance
 Laboratory Diagnosis:
Group B -Hemolytic
Streptococcus
Colony morphology
Grayish-white, mucoid, –
creamy, narrow zone of -
hemolysis
Presumptive Identification
tests
Catalase-negative –
Bacitracin-resistant –
 Laboratory Diagnosis:
Group B -Hemolytic
Streptococcus
Presumptive identification
tests
Bile-esculin-hydrolysis– –
negative
Does not grow in 6.5% NaCl –
CAMP-test–positive –

S. agalactiae shows the arrow-shaped

hemolysis near the staphylococcus
streak, showing a positive test for
CAMP factor
Identification Schema

Schema to differentiate Group A and B from

other b-hemolytic streptococci
 Streptococcus Group D
and Enterococcus Species
Members of the gut flora
Associated infections
Bacteremia –
Urinary tract infections –
Wound infections –
Endocarditis –
 Laboratory Diagnosis: Streptococcus
Group D and Enterococcus Species

Microscopic morphology
Cells tend to elongate –
Colony morphology
Most are non-hemolytic, –
although some may show
or, rarelyhemolysis
Possess Group D antigen –
Laboratory Diagnosis: Streptococcus Group D and
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
Identification Schema

Schema to differentiate Enterococcus and Group D

streptococci from other nonhemolytic streptococci
Other Streptococcal Species
Viridans group
Members of the normal oral and nasopharyngeal flora –
Includes those that lack the Lancefield group antigen –
Most are  hemolytic but also includes nonhemolytic species –
The most common cause of subacute bacterial endocarditis –
(SBE)