Microbio Lec 5 - Streptococcus

MICROBIOLOGY LECTURE 5 – Genus Streptococcus Notes from Lecture USTMED ’07 Sec C – AsM Generalities 1. 2. 3. 4.

Gram positive cocci that grow in pairs or chains Widely distributed in nature 27 recognized species 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. c. d. e.

7. 8.

b.

Alpha hemolysis showing partially hemolyzed RBC immediately surrounding the colonoy

Enterococcus faecalis(Streptococcus faecalis) (Group D) Streptococcus pneumoniae some of the oral streptococci

Streptococcus pyogenes (Group A) streptococcileading bacterial cause of pharyngitis and cellulitis; inciting factor of two important immunologic diseases: acute rheumatic fever and acute glomerunephritis 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 Streptococcus bovis – may also cause endocarditis

e.

9.

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

Streptococcus agalactiae (Group B)

Elaborates a variety of extracellular substances and enzymes Streptococci cause a wide variety of infections:

a.

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

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

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. b.

c.

clinical presentation(pyogenic,oral,enteric)

CLASSIFICATION OF STREPTOCOCCI

1.

a.

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

b.

c. d.

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

e.

2.

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

c.

Gamma hemolysis – no color change or lysis of red blood cells ; nonhemolytic Biochemical (physiological) properties • Sugar fermentation reactions • Tests for the presence of enzymes

• e.

Tests for susceptibility or resistance to certain chemical agents

d. e. f. 3.

c.

d. Beta hemolysis on 5% sheep blood agar 4. There is complete lysis of RBC surrounding the colony that can be seen macroscopically

main human pathogen associated with local or systemic invasion and poststreptococcal immunologic disorders PYR positive(hydrolysis of L-pyrrolidonyl-2napthylamide) usually susceptible to bacitracin

members of the normal flora of the female genital tract an important cause of neonatal sepsis and meningitis beta hemolytic hydrolyzes sodium hippurate gives a positive response to CAMP test

Groups C and G streptococci a. occur sometimes in the nasopharynx

b.

antigenic specificity of the capsular polysaccharide

contains the group A antigen beta hemolytic

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

b.

•

d.

Group A streptococcus – Streptococcus pyogenes

may cause sinusitis, bacteremia or endocarditis beta hemolytic identified by reactions with specific antisera for groups C or G

Group D streptococci

a.

enterococcal – Enterococcus faecium Enterococcus durans • Part of the normal enteric flora

• • •

Usually nonhemolytic; occasionally alpha hemolytic PYR positive Grow in the presence of bile

• • • •

Hydrolyze esculin (bile esculin positive) Grow in 6.5% NaCl resistant to penicillin G Some strains are vancomycin resistant

c.

usually alpha nonhemolytic

d.

part of the normal flora occasionally cause bacteremia or endocarditis can be found in brain abscesses and other infections

e. f.

b.

• • • • •

5.

• • • •

a.

Sometimes cause bacteremia in patients with colon carcinoma Nonhemolytic and PYR negative Grow in the presence of bile Hydrolyze esculin (bile esculinpositive) Do not grow in 6.5% NaCl Sensitive to penicillin

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

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. c.

9.

be

b.

grow only under anaerobic microaerophilic conditions variably produce hemolysis

or

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

Streptococcus anginosus or Streptococcus milleri, Streptococcus intermedius, Streptococcus constellatus a. part of the normal flora b. may be beta, alpha or nonhemolytic c. includes:

•

6.

may

11. Peptostreptococcus

Non-enterococcal – Streptococcus bovis • Part of the enteric flora • Occasionally cause endocarditis

•

hemolytic;

c. d.

colonies are not soluble bile(deoxycholate)

e.

important for the healthy state of the mucous membranes may reach the blood stream due to trauma

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

g.

a principal cause of abnormal heart valves

h.

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

endocarditis

on

10. Nutritionally

variant streptococci or pyridoxaldependent streptococci – Streptococcus defectives, Streptococcus adjacens a. require pyridoxal or cysteine for growth on blood agar

b.

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

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

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

f.

1.

grow as satellite colonies around colonies of staphylococci and other bacteria

ii.

iii. c.

o

anticomplimentary T or trypsin resistant protein o With M protein – an important epidemiological marker of group A strains o no relationship to virulence R protein

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

o ii.

Major adhesin for bacterial attachment to the epithelial cells of the pharynx and skin Lipoteichoic acid o Lipid moiety is implicated to binding to fibronectin. o Possibly mediates adherence to epithelial cells

Antigenic structure of Group B Streptococcus

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

-

-

3.

by

Group

Exotoxin B



c.

A 2.

a cysteine protease that rapidly destroys tissue and is produced by strains that cause necrotizing fasciitis Exotoxin A  May cause streptococcal toxic shock syndrome

Hemolysins

hyaluronidase(spreading factor)

-

2.

b.

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 Streptococcus (Streptococcus pyogenes)

1.

fever patient blanches the rash of a patient with scarlet fever

a.

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

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

Facilitates spreading of the microorganisms Antigenic – specific antibodies are found in the serum after infection with hyaluronidase producing organisms

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

 b.

streptodornase (streptococcal deoxyribonuclease)

Streptolysin O



-

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.



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 160200 units – suggests: • Recent infection with streptococci

•

Exotoxin C– classic erythrogenic toxin

 

causes the rash in scarlet fever



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)



Dick test – positive – erythema surrounding the injection site

Schultz Charlton reaction. Convalescent serum from a scarlet

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

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

Pathogenesis streptococci)

a. b.

c. d.

e. f.

of

Streptococcus

Persistently high antibody levels dueto an exaggerated immune response to an earlier exposure in a hypersensitive person agalactiae

(Group

B

is based on the ability of the organism to induce an inflammatory response no cytotoxic exotoxins are produced role of enzymes in the pathogenesis of infection is unknown – deoxyribonucleases, hyaluronidase, neuraminidase, proteases, hiuppurase and hemolysins no evidence for any immunologically induced disease has a polysaccharide capsule – antiphagocytic 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. 2.

Streptococcus pyogenes is the major cause of bacterial pharyngitis A disease of children 5-15 years

3. 4.

Spread by person to person by respiratory droplets

5.

Posterior pharynx erythematous with an exudate; cervical lymphadenopathy present

6.

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

Characterized by sore headache and nausea

throat,

fever,

malaise,

1. 2.

The primary site of the infection is usually the pharynx, with the distinctive rash resulting from an erythrogenic toxin produced by the streptococcus. 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. 2. 3. 1b. Cellulitis

1. 2.

3.

Cardinal features – erythema, swelling, heat and pain Erythema may be pink or red but lacks the intense, fiery red or salmon colored appearance of erysipelas.

desquamation of skin occurs 10-14 days after infection at sites that were erythematous during the initial phase 3a. Rheumatic fever

Initiated by infection through a small break in the skin

1.

Can invade the subcutaneous tissue and advance rapidly through lymphatics --à septicemia 1b. Erysipelas

2.

4.

1.

2.

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

Characterized by hypotension, diffuse erythroderma, hypoalbuminemia and multiorgan failure(kidney, lungs, liver, heart) serotypes implicated – M1,M3 or M18 due to the production of pyrogenic exotoxins – exotoxin A

3. 4.

most serious sequelae of hemolytic streptococcal infection because it results in damage to heart muscle and valves occurs 2 weeks after a group A streptococcal infection usually a pharyngitis results in a systemic inflammatory process involving the connective tissue, heart, joints, and CNS Characterized by fever, migratory polyarthritis, and carditis

5.

Due to an immunologic reaction between crossreacting 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

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

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

2a. Scarlet fever

1. 2.

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

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

6.

Diseases produced by Streptococcus agalactiae

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 streptococcus

beta hemolytic

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

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)

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

left(+) colorless; right (+) red color

Bacitracin susceptibility test

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.

IV.

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

4.

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

5. 6.

Nonterococcal Streptococcus bovis:penicillin G

3.

PYR Test

7.

-

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)

Viridans streptococci

-

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

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

- fin [email protected] [email protected]

-

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

-

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.

No zone of inhibition

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

-

mild - oral penicillin V

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

if allergic: erythromycin (azithromycin)

or

its

derivatives