STREPTOCOCCI

Microbiology Lecture 2 RS Module

Ashraf KhasawnehFaculty of Medicine

The Hashemite University

Streptococci

• Characters of Streptococci

– Gram positive round or lancet-shaped cocci

– 1µm in diameter

– Chains or pairs

– Usually capsulated

– Non motile

– Non spore forming

– Facultative anaerobes

– Catalase negative (Staphylococci are catalase positive)

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

Group A

S. pyogenes

Group B

S. agalactiae

Group C

S. equisimitis

Group D

Enterococcus

Other groups

(E-U)

Lanciefield

classification

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

Streptococci

-hemolysis -hemolysis -hemolysis

Hemolysis on BA

– -hemolysis

Partial hemolysis

Green discoloration around the colonies

e.g. non-groupable streptococci (S. pneumoniae & S. viridans)

– -hemolysis

Complete hemolysis

Clear zone of hemolysis around the colonies

e.g. Group A & B (S. pyogenes & S. agalactiae)

– -hemolysis

No lysis

e.g. Group D (Enterococcus spp)

Hemolysis on Blood agar

-hemolysis

-hemolysis

-hemolysis

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

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

– C5a peptidase

• Pyrogenic toxins that stimulate macrophages and helper T cells to release cytokines

• Streptolysins (pore forming cytotoxin)– Streptolysin O lyse red blood cells, white blood cells, and platelets

– Streptolysin S Oxygen stable

facilitate the spread of streptococci through tissues

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

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

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 Factor TestS. aureus

(Spingomyelinase C)Group B

Streptococcus

(CAMP Factor)

Group A

Streptococcus

Enhanced Zone of

Hemolysis

CAMP is an acronym for the authors of this test (Christie, Atkinson, Munch, Peterson). The CAMP

test takes advantage of the capacity of GBS-group B strep to produce this CAMP factor; most other

hemolytic streptococci do not produce CAMP factor. Enhances the ability of S. aureus to produce

Beta hemolysis

Differentiation between -hemolytic

streptococci

• The following definitive tests used to differentiate between S. pneumoniae & viridans streptococci

– Optochin Test

– Bile Solubility Test

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 susceptible

S. pneumoniae

Optochin resistant

S. viridans

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

Differentiation between -hemolytic streptococci

CAMP testBacitracin

sensitivity

Hemolysis

NegativeSusceptibleS. pyogenes

PositiveResistantS. agalactiae

Inulin

Fermentation

Bile

solubility

Optochin

sensitivity

Hemolysis

Not fermentSolubleSensitive (≥

14 mm)

S. pneumoniae

FermentInsolubleResistant

(≤13 mm)

Viridans strep

Differentiation between -hemolytic streptococci

Outline of differentiation between Gram-

Positive cocci

e.g. S. epidermidis

Clinical Infections

- Diverse group of acute suppurative (pus-forming) & nonsuppurative diseases

Skin and Soft tissue infections

A- Cellulitis

Streptococcal cellulitis is a spreading infection →bacteraemia

B- Erysipelas

Acute spreading skin lesion with lymphatic involvement

Marked oedema & erythema of skin; face and lower extremities, skin and subcutaneous tissues

C- Impetigo:

Contagious pyoderma with superficial yellow weeping lesions) - children

D- Necrotizing fasciitis: (a.k.a., “flesh-eating bacteria”):

Infection deep in subcutaneous tissues that spreads along fascial

planes, destroying muscle and fat; Initially cellulitis followed

by bullae (fluid filled blisters; bulla is singular), gangrene,

systemic toxicity, multiorgan failure and mortality in more than

50% of patients

E- Wound Infections

Erysipelas

NOTE:

erythema

bullae

Erysipelas is a type of

superficial cellulitis with

dermal lymphatic

involvement. Diagnosis

is clinical. Treatment is

with oral or IV

antibiotics.

Erysipelas is characterized

clinically by shiny, raised,

indurated, and tender

plaques with distinct

margins. High fever, chills,

and malaise frequently

accompany erysipelas.

There is also a bullous

form of erysipelas.

Flesh Eating Bacterium

Pictures courtesy of e-medicine

A patient out of the surgery room

after a flesh-eating bacteria

disease infected his leg.

An arm infection.

Streptococcus Pyogenes Clinical Infections

Upper Respiratory tract Infections

A- Tonsillitis / Pharyngitis

The commonest bacterial cause of tonsillitis

B- Scarlet fever

Streptococcal sore throat (Children < 10 years)

Incubation period is 2-3 days

Generalized erythema & Strawberry tongue

Scarlet fever: Complication of streptococcal pharyngitis when infecting strain is lysogenized; Frequently develop scarletina rash on upper chest spreading to extremities

C- Acute Otitis media & Sinusitis

STRAWBERRY TONGUE

tonsillitisOtitis media

Scarlet fever

Other Suppurative Diseases

•Puerperal & neonatal sepsis (child bed fever)

•Lymphangitis: Inflammation of lymphatic vessel(s)

•Pneumonia

Systemic Disease

•Streptococcal Toxic Shock Syndrome (TSS):Multisystem toxicity following soft tissue infection

progressing to shock and organ failure (not to be confused

with Staphylococcal Toxic Shock Syndrome where

hyperabsorbent tampons have been identified as an

important risk factor)

•Bacteremia

Group A Streptococcal Diseases

Nonsuppurative Sequelae

Post-infection complications of Group A streptococcal disease; Serious

complications in pre-antibiotic era; still important in developing countries

Acute rheumatic fever (ARF):

Inflammation of heart, joints, blood vessels, sub-cutaneous tissues

Rheumatic heart disease (RHD):

Chronic, progressive heart valve damage

Acute glomerulonephritis (AG):

Acute inflammation of renal (kidney) glomeruli

Nonsuppurative Sequelae of Acute Group A

Streptococcal Infection

Acute Rheumatic Fever (ARF)

ARF follows respiratory, not skin, infection

Inflammatory reaction characterized by arthritis, carditis, chorea (disorder of

CNS with involuntary spastic movements), erythema marginatum (skin redness

with defined margin), or subcutaneous nodules

Within 1-5 weeks following pharyngitis (Children 6-15 years)

• Epidemic pharyngitis: ARF in as many as 3%

• Sporadic pharyngitis: ARF in 1 per 1000

Morbidity & mortality linked to subsequent disease of heart valve (Rheumatic

Heart Disease)

Poorly understood pathogenesis with several proposed theories including cross-

reactivity of heart tissues & strep AGNs (M protein)

Nonsuppurative Sequelae of Acute Group A

Streptococcal Infection

Acute Glomerulonephritis

Follows either respiratory (pharyngitis) or cutaneous (pyoderma)

streptococcal infection

Associated with well-defined group of M-types

Incidence varies from <1% to 10-15%

Most often seen in children manifesting as dark, smokey urine with RBC's,

white blood cells, depressed serum complement, decreased glomerular

filtration rate (Oedema, Hypertension, Haematuria & Proteinuria)

Latent period: 1-2 weeks after skin infection and 2-3 weeks after pharyngitis

Granular accumulations of immunoglobulin due to deposition of immune

complexes within the kidney

Lab Identification of

S. pyogenes (Group A)

• Primary culture

• Domed grayish/opalescent

colonies

• Encapsulated cells produce

mucoid colonies

Beta-hemolytic• Zone several times greater than diameter of colony

Lab Identification of

S. pyogenes (Group A)

Catalase Negative: Differentiates from Staphylococcus

Bacitracin test: presumptively distinguishing between Group

A beta-hemolytic streptococci (bacitracin POS) and other beta-

hemolytic streptococci that are isolated from pharyngeal swabs

(95% sensitivity for Grp A strep)

When grown on blood agar, Group A streptococci are sensitive to

(killed by) the antibiotic bacitracin

Rapid Identification Tests:Based on extraction of Group A carbohydrate directly from

throat swabs

• ELISA, Coagglutination, Fluorescent Antibody

Streptococcus Pyogenes

Treatment All GAS are sensitive to penicillin G (no

resistance)

Pts allergic to penicillin, treat with Erythromycin or Azithromycin

Adequate treatment of strep pharyngitis within 10 days of onset prevent rheumatic fever but not glomerulonephritis.

Prevention of GAS

Penicillin prophylaxis with long acting preperations after primary attack of ARF (5-15 yrs)

No prophylaxis in case of AGN

Group B Streptococcus

Streptococcus agalactiae

Gram positive cocci in chains

Beta hemolytic

Catalase –ve

Bacitracin Resistant

CAMP test positive

Group B Streptococci:

Streptococcus agalactiae

•Major cause of neonatal sepsis

• 1 in 4 pregnant women are vaginal or

rectal carriers of GBS

• 50% of women transmit the disease to

infant (42-72%)

•Infect neonate during delivery

•1-2% colonized infants develop Group

B strep infection

•Induces pneumonia in the first 7 days

of life

• Later may induce septicemia ,

osteomyelitis and meningitis

Epidemiology of Neonatal Group B

Streptococcal Disease

Prevention /Detection

- 2002 WHO universal screening for GBS colonization by vaginal and rectal swab culture for pregnant women at 35-37 weeks gestation.

Lab confirmation

- Blood and CSF Culture , gram stain

- CAMP test

- Hippurate test (positive)

Treat GBS positive mothers with Penicillin G, Ampicillin or Eryrthromycin

Group D Streptococci (S.bovis)

– Gram-positive cocci,

– α, β or non-haemolytic on blood agar.

– Lancefield group D streptococci will grow on media containing bile and may be differentiated from other

streptococci by rapid hydrolysis of esculin in the presence of 40% bile. Bile esculine postive

• Endocarditis

Non-Lancefield Group Streptococci

Viridans StreptococciDental Caries: Streptococcus mutans

Streptococcus sanguis; Streptococcus

salivarius; Streptococcus mitis

Streptococus pneumoniae – # 1 cause of

community acquired pneumonia

Resistant to optochin

Streptococcus pneumoniae

• Commonly referred to as pneumococcus

• Formerly Diplococcus pneumoniae

• Diplococci lancet shaped

• Alpha hemolytic

• Optochin sensitive

S. pneumoniae

• Diplococcus

Virulence factors

• Colonization and migration factors

– IgA protease: help in colonization

– Pneumolysin- Pore forming toxin

• Tissue destruction

– Teichoic acid: colonization and dissemination processes

– Hydrogen peroxide: DNA damage and apoptosis in lung cells

– Pneumolysin- epithelial cell destruction

• Phagocytic survival

– Capsule – antigenic variation avoid phagocytosis -90 serotypes

• Autolysin: assist in pathogenesis due to its ability to break down the wall or lyse a portion of the invading pneumococci and release potentially lethal toxins into the cell

• S. pneumoniae is a leading cause of community pneumonia in all ages (particularly the very young and old), often after "damage" to the upper respiratory tract (e.g. following viral infection).

RUST COLOUR SPUTUM• Fever and chills. Difficulty breathing, cough

productive of purulent sputum and sometimes blood tinged, pleuritic chest pain

• It also causes middle ear infections (otitismedia).

• The organism often spreads causing bacteremiaand meningitis.

Lack structural damage to the lung

Complete resolution on recovery

Immunity

• Provided by antibody directed against the specific pneumococcal capsular type

• After ab binding, C3b is deposited and phagosytosis can take place.

• Diagnosis

• Gram stain: +ve diplococci

• Blood agar: alpha hemolysis, optochin sensitive

• Blood culture

• Detection of capsular antigen

Streptococcus pneumoniae

Diagnostic Laboratory Tests

• Optochin sensitivity (Taxo P disc)

Treatment

• Penicillin 1st choice

• First-generation cephalosporins are equally effective against infections caused by penicillin-susceptible S pneumoniae. All first-generation cephalosporins are equally effective against pneumococci;

• For empiric treatment of meningitis, third generation

cephalosporins may be administered in conjunction with vancomycin or rifampin.

• Azithromycin has activity against penicillin-susceptible strains of S pneumoniae.

Prevention

VACCINE

• Given the 90 different capsular types of pneumococci, a comprehensive vaccine based on polysaccharide alone is not feasible.

• Thus, vaccines based on a subgroup of highly prevalent types have been formulated. (PPV)

• The number of serotypes in the vaccine has increased from four in 1945, to 14 in the 1970s, and finally to the current 23-valent formulation (25 mg of each of serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F).

• These serotypes represent 85-90% of those that

cause invasive disease and the vaccine efficacy is estimated at 60% .

• T-cell independent; used in pts >2yrs• In 2000: PCV, 7-valent vaccine stimulte TH2

response and can be given at 2months. At 2 yrs give PPV

• Underutilization of the vaccine is so extensive that the pneumococcus remains the most common infectious agent leading to hospitalization in all age groups due to cost

• Further complicated by the fact that polysaccharides are not immunogenic in children under the age of 2 years where a significant amount of disease occurs

Immunization protocol

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324398/

• Polysaccharide antigens are postulated to stimulate splenic marginal zone B (MZB) cells, which do not mature until the second year of life [9]; therefore, the purified polysaccharide contained in 23vP, a T-independent antigen, is poorly immunogenic in young children. Chemical conjugation of pneumococcal polysaccharide to a carrier protein creates a T-dependent vaccine (pneumococcal conjugate vaccine [PCV]) that generates higher affinity antibodies, immunological memory, and induces responsiveness to booster doses of vaccine, resulting in a vaccine that is both immunogenic and highly effective from early infancy [10]. Because the splenic marginal zone is immature in early life, MZB cell responses are not present, and it is postulated that the conjugated polysaccharides in PCV are processed by the follicular origin (FO) B cells at that age [11]. Despite the immunological advantages of PCV in early childhood, both PCV7 (a 7-valent PCV) and 23vP induce similar antibody concentrations in adults [12], and it is therefore unclear whether the conjugate vaccine has any immunological advantage over 23vP or whether the same B-cell subsets are involved in the response.