Strep Pyogene and Strep Pnemonae

 Streptococcus organisms
Streptococus pneumoniae
and Streptococus pygene
Presentation
By
Ivan Kamulasi
2013-bmls-ft-008
School of allied health sciences
(c) ivankamulasi BMLS student,
CMLT 1

Streptococci
 Facultative anaerobes
 Gram-positive
 usually chains (sometimes pairs)
 catalase negative (compare staphylococci
are catalase positive)
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 Domain : Prokaryotes
 Kingdom : bacteria
 Phylum : Firmicutes
 Class : Bacilli
 Order : Lactobacillalees
 Family : Streptococcacae
 Genera : Streptococcus
 Species : S pyogenes and S pneumoniae
CMLT 3

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 The serology subdivision is based on the
difference in group specific polysaccharide
antigens in the cell wall.
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 Rebecca Lancefield working with various
streptococcal species discovered proteins in
the cell wall of β-streptococcus organism that
was unique to certain organisms. She later
said streptococci produce group specific
carbohydrates that can be identified using
group specific antiserum
CMLT 6

Grouped Streptococci
 A, B and D (frequent)
 C, G, F (less frequent)
Un grouped organisms
 S. pneumoniae (pneumonia)
 viridans streptococci (e.g. S. mutans ie dental
caries)
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 α (alpha)
◦ partial hemolysis
◦ green color
 β (beta)
◦ complete clearing
 γ (gamma)
- no lysis
8
White colonies
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 Groups A an B
◦β
 Group D
◦ α or γ
 S. pneumoniae and viridans
◦α
9
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 Is a spherical, gram positive bacterium
 It displays Streptoccocal group A antigen on
its cell wall.
 It’s a beta hemolytic
 Has an incubation period of 1-3 days
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 Class : Bacilli
 Species : S pyogenes
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 S pyogenes is widely distributed in nature
 Can be found in water, dust, vegetation, milk
products, and also as a commensal in the
upper respiratory tract.
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 Are gram positive and spherical
 They measure 0.7-0.9 um in diameter
 The cells occur in chains of varying lengths
and are best demonstrated in fluid cultures
where long chains are found
 They are capsulated in young cultures
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 Suitable culture media
 Blood agar, serum agar and MacConkey ( they
don’t grow on MacConkey due to salt present
in the media )
 Growth temperature ranges from 22-420c
with optimum at 37oC
 They measure 0.1-1mm in diameter after 24
hours of incubation
CMLT 14

 They are semi transparent, low convex,
desecrate and with matt or glossy surface ,
grey white or colorless, dry or shiny and
usually irregular outline on a Blood agar
 When freshly isolated, the colonies are
mucoid i.e heavily capsulated
 They are beta hemolytic on blood agar
CMLT 15

• Adherence to the epithelial cells
• Invasion into the epithelial cells - important for
persistent infections and invasion into deep
tissues
• Avoiding opsonization and phagocytosis;
• Producing enzymes and toxins
Streptococcus pyogenes Pathogenesis ( via
invasiveness and production of toxins
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Enzymes and toxins
Streptokinase (fibrinolysin)
Can lyse blood clots and may be responsible for the rapid spread
of the organism.
Used (IV injection) for treatment of pulmonary emboli, coronary
artery thrombosis and venous thrombosis.
Streptodornase (DNases A to D)
Decreases viscosity of DNA suspension.
Hyaluronidase (spreading factor):
Destroys connective tissue and aids in spreading infecting bacteria.
C5a peptidase
Prevents streptococci from activation of phagocytes and is
important for survival of S. pyogenes in tissue and blood.
Streptococcus pyogenes
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Hemolysins
Streptolysin O: O2-labile; causes hemolysis deep in blood
agar plates. ASO (antistreptolysin O) titer >160-200 units
suggests recent infection or exaggerated immune
response to an earlier respiratory infection. However, skin
infection does not induce ASO.
Streptolysin S: O2-stable. Causes b-hemolysis on the
surface of blood agar plates. Cell-bound, not antigenic.
Produced in the presence of serum. Kills phagocytes by
releasing the lysosomal contents after engulfment.
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Specimen
 Throat swab, pus, exudates, blood for culture
and serum for estimation of ASO titres
Micoscopy
Gram stain techniques;
They are gram positive cocci showing short or
long chains
Non motile and non sporing, however some
strain are capulated if prepared rom fresh
cultures
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 Suitable media
 Blood agar, chocolate agar, Macconkey
Incubate in 5-10% Co2 or aerobically at 37oc
On BA colonies are beta hemolytic measuring
0.5- 1 mm in diameter, grey white or colorless
with irregular shapes
On MacConkey the organisms show no growth
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 Lancefield grouping. This is done by rapid
and simple techniques such as streptex which
is a latex particle agglutination test.
Organism is Lancefield group A
 Serological test. The group antigens are
extracted using HCl and the antigens are then
tested with group specific antisera by one of
the following methods
 Precipitation test, Elisa, Fluorescent antibody
test (FAT), Slide agglutination test (SAT)
CMLT 22

 ASOT investigates post streptococcal diseases
such as rheumatic fever which give rise to
ASO level to 80-85% and this can be done by
ASO latex slide agglutination test which is a
screening test or ASO micro titration or tube
haemolysis test to titrate the antibody.
CMLT 23

 DNase B antibody to investigate acute
glomerulonephritis following a streptococcal skin
infection
 Antimicrobial sensitivity. Antibiotics with
activities against Streptococcus Pyogenes include
penicillin, erythromycin, Ampicillin, gentamycin,
ciprofloxacilin.
 Biochemical reactions
 They are Catalase negative, Bacitracin test
sensitive at a concentration of 0.05 units, Bile
solubility test negative, Camp test negative,
Litmus milk reduction test negative
CMLT 24

 Proper hygiene
 Vaccination
 Treatment of infected persons
CMLT 25

S. pyogenes can transiently colonize the oropharynx and skin.
Diseases are caused by recently acquired strains that can
establish an infection of the pharynx or skin.
S. pyogenes causes pharyngitis mainly in children of 5 to 15
years old.
The pathogen is spread mainly by respiratory droplets.
Crowding increases the opportunity for the pathogen to spread,
particularly during the winter months.
Soft tissue infections are preceded by skin colonization and the
organisms are introduced into the superficial or deep tissue
through a break in the skin.
Epidemiology
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Clinical Diseases
1. Local infection with S. pyogenes
Streptococcal sore throat (pharyngitis), and scarlet fever.
Streptococcal pyoderma (impetigo, local infection of
superficial layers of skin).
Strains that cause skin infections are different from those
that cause pharyngitis.
Additional notes
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2. Invasion by S. pyogenes
Invasion from respiratory tract: otitis media, sinusitis,
pneumonia, meningitis, osteomyelitis, and arthritis.
Invasion from skin: erysipelas, cellulitis, and necrotizing
fasciitis. Diffuse and rapidly spreading infection that
extends along lymphatic pathways with only minimal local
suppuration.
Sepsis (streptococcal toxic shock syndrome, STSS):
the organism is introduced into the subcutaneous tissue
through a break in the skin cellulitis necrotizing
fasciitis systemic toxicity, multiple organ failure, and
death (mortality > 40%).
CMLT 28

3. Poststreptococcal diseases (occurs 1-4 weeks after
acute S. pyogenes infection, hypersensitivity responses)
Rheumatic fever: most commonly preceded by infection of the
respiratory tract. Inflammation of heart (pancarditis), joints,
blood vessels, and subcutaneous tissue. Results from cross
reactivity of anti-M protein Ab and the human heart tissue.
Acute glomerulonephritis: preceded by infection of the skin
(more commonly) or the respiratory tract. Symptoms: edema,
hypertension, hematuria, and proteinuria. Initiated by Ag-Ab
complexes on the glomerular basement membrane.
* Rheumatic fever can be reactivated by recurrent streptococcal
infections, whereas nephritis does not.
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Streptococcus
pneumonae
CMLT 30

 leading cause of pneumonia
◦ particularly young and old
 bacteremia
 meningitis
 middle ear infections (otitis media) -
children
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 Class : Bacilli
 Species : S pneumoniae
CMLT 32

member normal flora, nasopharynx of 10 – 30 %
healthy individuals
replication and spread after damage to upper
respiratory tract (e.g. after the flu)
CMLT 33

MORPHOLOGY:
 Pneumococci are Gram
positive small(1μm),
slightly elongated cocci,
with one end broad &
other end pointed,
presenting a flame
shaped or lanceolate
appearance.
 They occur in pairs,
with the broad ends
opposing each other.
 They are capsulated &
the capsule encloses
each pair.
 They are nonmotile &
nonsporing.
CMLT 34

CULTURE & CULTURAL CHARACTERISTICS:
 They grow only in enriched media.
 They are aerobes & facultative anaerobes
 The optimum temperature being 37ºC & pH 7.8.
 Growth is improved by 5-10% CO2.
 On BA, the colonies are small, smooth and
transparent low convex, may be white, tiny and
become flattened and measure 1mm in diametre
(doom shape)
CMLT 35

Media used: Blood agar
Colony morphology: On blood agar, after
incubation for 18 hours, the colonies are small,
dome shaped & glistening, with an area of
α-haemolysis.
On further incubation the colonies
become flat with raised edges & central
umbonation called as Draughtsman or carrom
coin appearance.
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CMLT 37

PATHOGENICITY:
Source of infection:
i) Endogenous- from the colonized area.
ii) Exogenous- patients or carriers.
Mode of infection: By inhalation.
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Antigenic structure:
1. Capsular polysaccharide:
 It is the most important antigen & type
specific.
 Since it diffuses into infective tissue & culture
medium it is called as specific soluble
substance(SSS).
 Pneumococci are classified into types based
on the nature of capsular polysaccharide &
more than 90 serotypes are recognised &
named 1,2,3…...
CMLT 39

2. M protein: It is not associated with virulence.
3. ‘C’ Carbohydrate antigen:
- It is present in all pneumococci so species
specific.
- Virulance factors
1. Capsule: It is antiphagocytic.
2. Pneumolysin: It is a membrane damaging
toxin has cytotoxic and complement
activating properties.
CMLT 40

Antigenic structure & virulence factors of S.pneumoniae(c) ivankamulasi BMLS student,
CMLT 41

D. Determinants of Pathogenicity
Toxins
a) Pneumolysin O (Ply)
 A 53-kDa protein that is cytolytic to eukaryotic cells
that have cholesterol as a component of their cell
membranes particularly the respiratory epithelium;
also activates complement
b) Autolysin (LytA)
 Causes lysis of pneumococci in the presence of
surface-active agents or antimicrobials that inhibit cell
wall synthesis
 Release toxic proteases
Cell wall components
 Teichoic acid & peptidoglycan beneath the capsular
polysaccharide
(c) ivankamulasi BMLS
student, CMLT 42

D. Determinants of Pathogenicity
Hydrogen peroxide –
• causes damage to host cells (can cause apoptosis
in neuronal cells during meningitis) and has
bactericidal effects against competing bacteria
(Haemophilus influenzae, Neisseria meningitidis,
Staphylococcus aureus)
Pili –
• hair-like structures that extend from the surface
• contributes to colonization of upper respiratory
tract and increase the formation of large amounts
of TNF by the immune system during sepsis,
raising the possibility of septic shock
student, CMLT 43

Mechanism of Pathogenesis:
Entry of pneumococci into nasopharynx
Colonization of nasopharynx
May cause infection of the middle ear,
paranasal
sinuses & respiratory tract by direct spread
Infection of meninges can also occur, by
contiguity or
through blood
Enters blood causing bacteremia, which may
also
lead to disseminated infections as in the
heart,
peritoneum or joint(c) ivankamulasi BMLS student,
CMLT 44

LABORATORY DIAGNOSIS:
Specimens to be collected:
 Sputum,
 CSF,
 Blood,
 Synovial fluid,
 In children laryngeal swab can be taken if sputum
can not be collected.
CMLT 45

1.Direct
microscopy:
Gram stained
smears reveals
Gram positive
lanceolate shaped
diplococci with
numerous pus
cells.
CMLT 46

3. Antigen detection: Capsular polysaccharide
antigen in blood, CSF & urine can detected by
 Passive latex agglutination,
 Counter immunoelectrophoresis,
 Coaggutination.
CMLT 47

Counter immunoelectrophoresis
CMLT 48

4. Culture:
a) Media used:
Blood agar
b) Colony morphology:
Are small, smooth and transparent, low convex,
may be white, tiny and become flattened or
depressed centrally showing draughts-man’s
shape and measure up to 1mm in diameter
(doom shape)
c) Indirect Gram’s smear:
Smears are examined
from the culture plate
and reveals Gram
positive lanceolate
shaped diplococci.
CMLT 49

 Streptococcus pneumoniae is a fermentative
aerotolerant anaerobe.
 It is usually cultured in media that contain blood.
On blood agar, colonies characteristically
produce a zone of alpha (green) hemolysis, which
differentiates S. pneumoniae from the group A
(beta hemolytic) streptococcus, but not from
commensal alpha hemolytic (viridans)
streptococci which are co-inhabitants of the
upper respiratory tract.
 Special tests such as inulin fermentation, bile
solubility, and optochin (an antibiotic) sensitivity
must be routinely employed to differentiate the
pneumococcus from Streptococcus viridans.
CMLT 50

d) Capsular swelling reaction: Positive.
It is done by mixing the suspension of colonies
from the culture plate and a loopful of type
specific antiserum & a drop of methylene blue
solution on a slide.
e) Biochemical reactions:
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OPTOCHIN TEST (ethylhydrocupreine HCl)
• Inhibits growth of pneumococci but not viridans
student, CMLT 52
Optochin positive Optochin negative

f) Optochin sensitivity: Sensitive.
CMLT 53

BILE SOLUBILITY TEST
• Bile or bile salts (surface-active agents)
activate an autolytic AMIDASE which
cleaves the bond between alanine &
muramic acid in the peptidoglycan
resulting in lysis of microorganism
• Amidase is present in pneumococcus
but not in viridans streptococci
student, CMLT 54

(Neufeld) QUELLUNG (capsular
precipitation) REACTION
• Most rapid & most useful: identifies & specifies type
of pneumococci in sputum, spinal fluid, exudates, or
culture
• Pneumococcal specimen mixed with (polyvalent)
antipneumococcal serum & methylene blue:
 Positive result: refractile & swollen capsules on oil
immersion
student, CMLT 55

2. Quellung( capsular swelling ) reaction:
 It is described by Neufeld.
 On a slide the sputum is mixed with type specific
antiserum against capsular antigen & a loopful of
methylene blue solution. The capsule becomes
swollen & refractile.
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5. Animal inoculation: From specimens where
organisms are expected to be scanty, isolation
may be obtained by intraperitoneal inoculation
in mice.
6. Serology: Antibodies can be demonstrated by
agglutination & precipitation test.
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Catalase test
 Facultative anaerobe
 Catalase-negative:
accumulation of hydrogen
peroxide kills microorganism
in culture medium
student, CMLT 58

• MANAGEMENT
– CHEMOTHERAPY:
• Based on sensitivity teating
• DOC: IM PCN G (uncomplicated pneumonia) OR
oral PCN V (milder URTI)
• PCN-allergic alternatives: cephalosporin or
erythromycin (pneumonia),chloramphenicol
(meningitis), quinolones
– PREVENTIVE:
• Pneumococcal conjugate vaccine for high-risk cases
student, CMLT 59

 Reservoir Human carriers
 Transmission Respiratory
"Autoinoculation“
 Temporal pattern Winter and early spring
 Communicability Unknown
Probably as long as
organism in respiratory
secretions
student, CMLT 60

 Outbreaks uncommon
 Generally occur crowded
environments (jails, nursing
homes)
 Persons with invasive
disease often have
underlying illness
 May have high fatality rate
student, CMLT 61

student, CMLT 62

Invasive Pneumococcal Disease
Incidence by Age Group
CMLT 63
0
50
100
150
200
250
<1 1 2 3 4 5-17 18-34 35-49 50-64 65+
Age Group (Yrs)
Rate
Rate per 100,000 population. Active Bacterial Core Surveillance/EIP Network

Commentary
Additions
Subtractions
Recommendations are all welcome
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thank for your time and listening
A blessed week please!!!!
CMLT 65

Strep Pyogene and Strep Pnemonae

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