Microbio Lec 5 - Streptococcus

MICROBIOLOGY LECTURE 5 – Genus Streptococcus Notes from Lecture

USTMED ’07 Sec C – AsM Generalities

1. Gram positive cocci that grow in pairs or chains 2. Widely distributed in nature

3. 27 recognized species

4. Some are members of the normal human flora

5.

Others are associated with important human disease attributable to infection by streptococci and to sensitization to them

6.

The most important of the human pathogens: a. Streptococcus pyogenes (Group A)

b.

Streptococcus agalactiae (Group B)

c.

Enterococcus faecalis(Streptococcus faecalis) (Group D)

d. Streptococcus pneumoniae e. some of the oral streptococci

7.

Elaborates a variety of extracellular substances and enzymes

8. Streptococci cause a wide variety of infections:

a.

Streptococcus pyogenes (Group A) streptococci- leading bacterial cause of pharyngitis and cellulitis; inciting factor of two important immunologic diseases: acute rheumatic fever and acute glomerunephritis

b. Streptococcus agalactiae (Group B streptococci)- leading cause of neonatal sepsis and meningitis

c.

Enterococcus faecalis – important cause of hospital acquired urinary tract infections and endocarditis

d.

Viridans group streptococci – most common cause of endocarditis

e. Streptococcus bovis – may also cause endocarditis

9.

Most species are facultative anaerobes; maybe strictly anaerobic to capnophilic

10.

Have complex nutritional requirements necessitating blood or serum enriched medium for isolation

11.

Carbohydrates are fermented with the production of lactic acid but not gas

12.

Catalase negative 13. Can be classified as to:

a.

clinical presentation(pyogenic,oral,enteric)

b.

serological properties (Lancefield grouping A-H, K-V) based on the antigenic composition of cell wall carbohydrates

• serogroups A,B,C,D and G – most commonly found associated with human disease

c.

Hemolytic patterns in blood agar plates

•

Beta hemolysis – clear zone of hemolysis around the colony as a result of complete lysis of the red blood cells

•

Alpha hemolysis – zone of partial hemolysis with a greenish discoloration of the medium

•

Gamma hemolysis – no color change or lysis of red blood cells ; nonhemolytic d. Biochemical (physiological) properties

• Sugar fermentation reactions • Tests for the presence of enzymes

•

Tests for susceptibility or resistance to certain chemical agents

e.

antigenic specificity of the capsular polysaccharide Beta hemolysis on 5% sheep blood agar

There is complete lysis of RBC surrounding the colony that can be seen macroscopically

There is a clear colorless zone around a streptococcal colony in which the RBC have undergone complete destruction

Alpha hemolysis on 5% sheep blood agar

an indistinct zone of partial lysis of red blood cells causing a green to greenish brown discoloration of the medium immediately surrounding the colony

Alpha hemolysis showing partially hemolyzed RBC immediately surrounding the colonoy

Gamma hemolysis on 5% sheep blood agar Gamma denotes lack of

hemolysis; the RBC surrounding the colonies are intact

Macroscopically there is no apparent hemolytic activity or discoloration produced by the colony

CLASSIFICATION OF STREPTOCOCCI

1. Group A streptococcus – Streptococcus pyogenes

a.

contains the group A antigen b. beta hemolytic

c.

main human pathogen associated with local or systemic invasion and poststreptococcal immunologic disorders

d. PYR positive(hydrolysis of L-pyrrolidonyl-2-napthylamide)

e. usually susceptible to bacitracin

2.

Group B streptococcus – Streptococcus agalactiae a. contains the group B antigen

b.

members of the normal flora of the female genital tract

c. an important cause of neonatal sepsis and meningitis

d. beta hemolytic

e. hydrolyzes sodium hippurate

f. gives a positive response to CAMP test 3. Groups C and G streptococci

a. occur sometimes in the nasopharynx

b.

may cause sinusitis, bacteremia or endocarditis c. beta hemolytic

d.

identified by reactions with specific antisera for groups C or G

4. Group D streptococci

a.

enterococcal – Enterococcus faecium

Enterococcus durans • Part of the normal enteric flora

•

Usually nonhemolytic; occasionally

alpha hemolytic • PYR positive

• Hydrolyze esculin (bile esculin positive)

• Grow in 6.5% NaCl • resistant to penicillin G

• Some strains are vancomycin resistant

b. Non-enterococcal – Streptococcus bovis • Part of the enteric flora

• Occasionally cause endocarditis

•

Sometimes cause bacteremia in

patients with colon carcinoma • Nonhemolytic and PYR negative • Grow in the presence of bile

• Hydrolyze esculin (bile esculin-positive)

• Do not grow in 6.5% NaCl • Sensitive to penicillin

5.

Streptococcus anginosus or Streptococcus milleri, Streptococcus intermedius, Streptococcus constellatus

a. part of the normal flora

b. may be beta, alpha or nonhemolytic c. includes:

•

Beta hemolytic streptococci that form minute colonies (<0.5 mm in diameter) and react with groups A,C or G antisera

• All beta hemolytic group F streptococci

• Voges Proskauer test positive

• Those that are group A are PYR negative

• May be classified as Viridans streptococci

6. Group N streptococci

a. rarely found in human disease states

b.

produce normal coagulation (souring ) of milk

7. Groups E,F,G,H and K-U streptococci - occur primarily in animals 8. Streptococcus pneumoniae

a. alpha hemolytic

b.

growth inhibited by optochin (ethylhydrocupreine hydrochloride) c. colonies are bile soluble

9.

Viridans streptococci – Streptococcus mitis, Streptococcus salivarius, Streptococcus sanguis (Group H), Streptococcus mutans

a. typically alpha hemolytic ; may be nonhemolytic

b. growth not inhibited by optochin

c.

colonies are not soluble bile(deoxycholate)

d.

most prevalent members of the normal flora of the upper respiratory tract

e.

important for the healthy state of the mucous membranes

f. may reach the blood stream due to trauma

g.

a principal cause of endocarditis on abnormal heart valves

h.

Streptococcus mutans – synthesizes large polysaccharides (dextrans and levans) from sucrose which may lead to dental caries

10.

Nutritionally variant streptococci or pyridoxal-dependent streptococci – Streptococcus defectives, Streptococcus adjacens

a. require pyridoxal or cysteine for growth on blood agar

b.

grow as satellite colonies around colonies of staphylococci and other bacteria

c.

usually alpha hemolytic; may be nonhemolytic

d.

part of the normal flora

e. occasionally cause bacteremia or endocarditis

f. can be found in brain abscesses and other infections

11. Peptostreptococcus

a.

grow only under anaerobic or microaerophilic conditions

b. variably produce hemolysis

c.

part of the normal flora of the mouth, upper respiratory tract, bowel and female genital tract

d.

often participate in mixed anaerobic infections in the abdomen, pelvis, lung or brain

Antigenic Structure of Group A Streptococcus

1.

Capsule

- hyaluronic acid; nonimmunogenic; antiphagocytic 2. Cell wall

a.

group specific carbohydrates of group A – is a dimer of N-acetylglucosamine and rhamnose

b. type specific protein antigens i. M protein

o a major antigen associated with virulent streptococci

o

located at the end of the hairlike fimbriae that are anchored in the cell wall and extending through the capsule

o antiphagocytic

o

anticomplimentary ii. T or trypsin resistant protein

o

With M protein – an important epidemiological marker of group A strains o no relationship to virulence

iii. R protein c. Other surface antigens

i. F protein or fibronectin binding protein o Has a receptor for fibronectin

o

Major adhesin for bacterial attachment to the epithelial cells of the pharynx and skin ii. Lipoteichoic acid

o

Lipid moiety is implicated to binding to fibronectin.

o Possibly mediates adherence to epithelial cells

PATHOGENESIS

Group A streptococci (Streptococcus pyogenes) cause disease by three mechanisms:

-

pyogenic inflammation – induced locally at the site of the organisms in tissue

-

exotoxin production – can cause widespread systemic symptoms in areas of the body where there are no organisms

-

immunologic – occurs when antibody against a component of the organism cross-reacts with normal tissue or forms immune complexes that damage normal tissue

Inflammation-related enzymes produced by Group A Streptococcus (Streptococcus pyogenes)

1.

hyaluronidase(spreading factor)

-

degrades hyaluronic acid which is the ground substance of the connective tissue

-

Facilitates spreading of the microorganisms

-

Antigenic – specific antibodies are found in the serum after infection with hyaluronidase producing organisms

2. streptokinase(fibrinolysin)

-

Transforms plasminogen of human plasma into plasmin, an active proteolytic enzyme that digests fibrin and other proteins

-

Given intravenously for treatment of pulmonary emboli and of coronary artery and venous thromboses 3. streptodornase (streptococcal deoxyribonuclease)

-

Depolymerizes DNA in exudates or necrotic tissue

-

With streptokinase – used in enzymatic debridement; Helps liquefy exudates and facilitates removal of pus and necrotic tissue

-

Antibody to Dnase develops after streptococcal skin infection (normal limit –100 units)

Toxins and hemolysins produced by Group A streptococci (Streptococcus pyogenes)

1. Streptococcal pyrogenic toxins - Three antigenically distinct toxins

a.

Exotoxin C– classic erythrogenic toxin



causes the rash in scarlet fever



Produced only by strains lysogenized by a bacteriophage carrying the gene for the toxin



Dick test – The injection of a skin test dose of erythrogenic toxin gives a positive result (an erythematous reaction in the skin of nonimmune persons who lack antitoxins)



Schultz Charlton reaction – antitoxin injected into the skin of a patient with scarlet fever causes localized blanching as a result of neutralization of erythrogenic toxin

Dick test – positive – erythema surrounding the injection site

Schultz Charlton reaction. Convalescent serum from a scarlet

fever patient blanches the rash of a patient with scarlet fever

b. Exotoxin B



a cysteine protease that rapidly destroys tissue and is produced by strains that cause necrotizing fasciitis

c. Exotoxin A

 May cause streptococcal toxic shock syndrome

2. Hemolysins

a. Streptolysin S



oxygen stable, nonimmunogenic cell bound hemolysin capable of lysing eryhtrocytes, leukocytes and platelets



stimulate release of lysosomal contents after engulfment



responsible for the hemolytic zones around streptococcal colonies growing on the surface of blood agar

 not antigenic

b. Streptolysin O



a protein that is hemolytically active in the reduced state  responsible for the hemolysis

seen when growth is in cuts deep into the medium in blood agar



antigenic – antibodies are formed against streptolysin O following infection with streptococci that produce streptolysin O

 ASO serum titer in excess of 160-200 units – suggests:

• Recent infection with streptococci

•

Persistently high antibody levels dueto an exaggerated immune response to an earlier

exposure in a

hypersensitive person

Pathogenesis of Streptococcus agalactiae (Group B streptococci)

a.

is based on the ability of the organism to induce an inflammatory response

b. no cytotoxic exotoxins are produced

c.

role of enzymes in the pathogenesis of infection is unknown – deoxyribonucleases, hyaluronidase, neuraminidase, proteases, hiuppurase and hemolysins

d. no evidence for any immunologically induced disease

e.

has a polysaccharide capsule – antiphagocytic f. anticapsular antibody is protective

CLINICAL FINDINGS

Types of diseases produced by Streptococcus pyogenes 1. Pyogenic diseases

a. pharyngitis

b. cellulitis and erysipelas c. impetigo(pyoderma) 2. Toxigenic diseases

a. scarlet fever b. toxic shock syndrome 3. Immunologic diseases

a. rheumatic fever b. acute glomerunephritis

1a. Pharyngitis

1.

Streptococcus pyogenes is the major cause of bacterial pharyngitis

2. A disease of children 5-15 years

3.

Spread by person to person by respiratory droplets

4.

Characterized by sore throat, fever, malaise, headache and nausea

5.

Posterior pharynx erythematous with an exudate; cervical lymphadenopathy present

6.

Can result to complications (tonsillar abscesses, mastoiditis, septicemia, osteomyelitis, rheumatic fever)

1b. Cellulitis

1.

Cardinal features – erythema, swelling, heat and pain

2. Erythema may be pink or red but lacks the intense, fiery red or

salmon colored

appearance of

erysipelas.

3.

Initiated by infection through a small break in the skin

4.

Can invade the subcutaneous tissue and advance rapidly through

lymphatics --à

septicemia 1b. Erysipelas

1.

Characteristic appearance – bright red or salmon red painful confluent erythema in a “butterfly” distribution involving the nasal eminence, cheeks, and nose with abrupt borders along the nasolabial folds

2.

Erythema increases over a course of 3-6 days and usually resolves in 7-10 days

3.

Erysipelas usually occur on the face, although any skin surface such as the leg, can be affected.

4.

Note the sharp line of demarcation and bright red color, features that distinguish it from cellulitis 1c. Impetigo

1. A superficial infection that usually begins as small vesicles

progressing to weeping lesions with amber crust and slightly cloudy purulent exudate. 2. Serotypes implicated – M

types 2,49, 55 & 57 3. May result to nephritis

as a complication

2a. Scarlet fever

1.

The primary site of the infection is usually the pharynx, with the distinctive rash resulting from an erythrogenic toxin produced by the streptococcus. 2. The rash appears within 2 days after the onset of the

sore throat and disappears in 6-10 days. Left – scarlet fever

Right – measles

2b. Streptococcal toxic shock syndrome (also called toxic shock like syndrome)

1.

Characterized by hypotension, diffuse erythroderma, hypoalbuminemia and multiorgan failure(kidney, lungs, liver, heart)

2. serotypes implicated – M1,M3 or M18

3. due to the production of pyrogenic exotoxins – exotoxin A

desquamation of skin occurs 10-14 days after infection at sites that were erythematous during the initial phase

3a. Rheumatic fever

1.

most serious sequelae of hemolytic streptococcal infection because it results in damage to heart muscle and valves

2. occurs 2 weeks after a group A streptococcal infection usually a pharyngitis

3.

results in a systemic inflammatory process involving the connective tissue, heart, joints, and CNS 4. Characterized by fever, migratory polyarthritis, and

carditis

5.

Due to an immunologic reaction between cross-reacting antibodies to certain streptococcal M proteins and antigens of joint and heart tissue

6.

Treat promptly with penicillin which is continued prophylactically to prevent recurrence and increased damage

Revised Jones Criteria for the Diagnosis of Rheumatic Fever The diagnosis of rheumatic fever is highly likely if supported by evidence of a preceding group A streptococcal infection and the presence of two major manifestations or one major and two minor manifestations.

Supporting evidence of antecedent group A streptococcal infection

Positive throat culture

Positive streptococcal antigen test

Elevated or rising streptococcal antibody titer Major Manifestations Carditis Polyarthritis Chorea Erythema marginatum Subcutaneous nodules Minor manifestations

Clinical findings: arthalgia, fever Laboratory findings

Elevated acute phase reactants (erythrocyte sedimentation rate, C-reactive protein)

Prolonged PR interval on elevtrocardiography 3b. Acute glomerulonephritis

1.

Typically occurs 2-3 weeks after streptococcal skin infections with M types 2,4,12 or 49 (most frequent) 2. More frequent after skin infections than after

3.

Characterized by hypertension, edema of the face(especially periorbital edema) and ankles, & “smoky” urine

4.

Complete recovery; reinfection with streptococci rarely leads to recurrence.

5.

Initiated by deposition of soluble streptococcal antigen-antibody complexes and complement on the glomerular basement membrane – lumpy-bumpy pattern on immunofluorescence

6. Can be prevented by early eradication of nephritogenic streptococci from skin colonization sites

Diseases produced by Streptococcus agalactiae

Neonatal Group B streptococcal disease LABORATORY DIAGNOSIS

I. Streptococcus pyogenes 1. Microscopy – Gram

stain

Gram stain of streptococci in a positive broth culture Gram positive cocci in chains

2.

Cultures Colonies of group A streptococci on 5% sheep blood agar small colonies with a wide zone of beta hemolysis

Group A Streptococcus

Streptococcus selective agar – contains sulfamethoxazole And trimethoprim which inhibits the growth of nongroup A betahemolytic streptococci, staphylococci, viridans Streptococci and gram negative bacilli

Left – blood agar plate; right – streptococcus selective agar PYR Test

The presence of an aminopeptidase enzyme that degrades the substrate is a 10 minute presumptive test for Group A streptococci (beta hemolytic) and Aerococcus, Enterococcus and Gemella(alpha or nonhemolytic)

left(+) colorless; right (+) red color Bacitracin susceptibility test

An alternative to PYR test for the presumptive identification of Group A beta hemolytic streptococci 0.04 units of bacitracin disk is placed on an inoculum of the microorganism on sheep blood agar

Positive test – zone of inhibition

II.

Streptococcus agalactiae - Group B beta hemolytic streptococcus

1. Microscopy

- Gram stain of vaginal secretions

Gram positive cocci in pairs,suggestive of Streptococcus

agalactiae which colonize the genitourinary tract of women

2. culture

Colonies of Group B streptococcus on 5% sheep blood agar

Colonies are larger than other beta hemolytic streptococci. Hemolytic zone surrounding the colony is smaller.

Tests

1. Hippurate hydrolysis test

Incubate a suspension of the microorganism for 2 hours at 35 C in a hippurate solution. Add ninhydrin(indicator) Hydrolysis of sodium hippurate leads to the formation of glycine and sodium benzoate. Deamination of glycine –purple color

2.

CAMP test (Christie,Atkins, Munich-Peterson) - An alternative to hippurate hydrolysis

Demonstrates the arrowhead shaped enhancement of beta hemolysis that occurs when the hemolytic beta toxin producedby Staphylococcus aureus acts synergistically with the CAMP factor

III. Group D streptococci 1. enterococcus Colonies of Enterococcus spp. on 5% sheep blood agar Colonies are raised, white to gray white ranging from 0.5 to 1.5 mm. In size and are usually nonhemolytic

Tests

1. Bile esculin and 6.5% NaCl

-

Bile esculin slant(left) – indicates that the microorganism can grow in the presence of bile and hydrolyze esculin.

2.

6.5% NaCl broth(right)- growth is indicated by turbidity and change in the indicator from pink to yellow

3. PYR Test

-

The presence of an aminopeptidase enzyme that degrades the substrate PYR(L-pyrrolidonyl-B napthylamide) is a 10 minute presumptive test for group A streptococci(beta Hemolytic) and Aerococcus, Enterococcus and Gemella(alpha or nonhemolytic)

IV. Viridans streptococci

-

Gram stain of a viridans streptococcus species in Blood culture broth appear in long chains especially when recovered from a blood culture broth

-

Optochin susceptibility test. A paper disk containing optochin (ethylhydrocupreine Hydrochloride) is applied to the surface of an inoculated 5% sheep blood agar plate.

No zone of inhibition

-

Identification of viridans streptococci with conventional biochemical reactions. Definitive identification requires several substrates including Bile esculin, arginine decarboxylase, 6.5% NaCl, lactose, Mannitol, raffinose, sorbitol, arabinose, inulin, sucrose and Esculin.

TREATMENT

1. All group A streptococci are susceptible to penicillin G.

-

mild - oral penicillin V

-

if allergic: erythromycin or its derivatives (azithromycin)

2.

Endocarditis caused by Viridans streptococci is curable by prolonged penicillin treatment

3.

Enterococcal endocarditis – eradicated only by a penicillin or vancomycin combined with an aminoglycoside

4.

Vancomycin resistant enterococci – linezolid (Zyvox) and quinupristin/dalforpristin(Synercid)

5.

Nonterococcal Streptococcus bovis:penicillin G

6.

Group B streptococcal infections- penicillin G or ampicillin in combination with an aminoglycoside 7. Peptostreptococci – penicillin G

PREVENTION

1.

Rheumatic fever can be prevented by prompt treatment of Group A streptococcal pharyngitis with penicillin.

2.

Penicillin prophylaxis for acute rheumatic fever patients to prevent recurrence of the disease; not needed in acute glomerulonephritis

3.

In patients with damaged heart valves who undergo invasive dental procedures, endocarditis can be prevented by using amoxicillin perioperatively.

4.

In patients with damaged heart valves who undergo gastrointestinal or urinary tract procedures, endocarditis caused by enterococcus can be prevented by using ampicillin and gentamicin perioperatively.

5.

Neonatal sepsis caused by group B streptococci can be prevented by administration of parenteral ampicillin perinatally to women who experience prolonged (longer than 18 hours) rupture of membranes, whose labor begins before 37 weeks gestation or who have a fever at the time of labor.

- fin -

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