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

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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

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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

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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

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CMLT

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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Domain : Prokaryotes

Kingdom : bacteria

Phylum : Firmicutes

Class : Bacilli

Order : Lactobacillalees

Family : Streptococcacae

Genera : Streptococcus

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

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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

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• 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)

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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.

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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

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Proper hygiene

Vaccination

Treatment of infected persons

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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%).

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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

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leading cause of pneumonia◦ particularly young and old

bacteremia

meningitis

middle ear infections (otitis media) -children

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Domain : Prokaryotes

Kingdom : bacteria

Phylum : Firmicutes

Class : Bacilli

Order : Lactobacillalees

Family : Streptococcacae

Genera : Streptococcus

Species : S pneumoniae

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member normal flora, nasopharynx of 10 – 30 % healthy individuals

replication and spread after damage to upper respiratory tract (e.g. after the flu)

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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.

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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)

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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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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…...

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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.

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

D. Determinants of PathogenicityToxins

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

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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

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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 alsolead 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.

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1.Direct microscopy:

Gram stainedsmears revealsGram positivelanceolate shapeddiplococci withnumerous pus

cells.

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3. Antigen detection: Capsular polysaccharide

antigen in blood, CSF & urine can detected by

Passive latex agglutination,

Counter immunoelectrophoresis,

Coaggutination.

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Counter immunoelectrophoresis(c) ivankamulasi BMLS student,

CMLT 48

4. Culture:a) Media used:Blood agarb) 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.(c) ivankamulasi BMLS student,

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.

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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

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Optochin positive Optochin negative

f) Optochin sensitivity: Sensitive.

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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

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(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

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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

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• 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

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Reservoir Human carriers

Transmission Respiratory"Autoinoculation“

Temporal pattern Winter and early spring

Communicability UnknownProbably as long asorganism in respiratorysecretions

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Outbreaks uncommonGenerally occur crowded environments (jails, nursing homes)

Persons with invasive disease often have underlying illness

May have high fatality rate(c) ivankamulasi BMLS

student, CMLT 61

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student, CMLT 62

Invasive Pneumococcal DiseaseIncidence by Age Group

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0

50

100

150

200

250

<1 1 2 3 4 5-17 18-34 35-49 50-64 65+

Age Group (Yrs)

Ra

te

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!!!!

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